Methods for arraying, soaking solutions, and selecting soaking conditions for small molecules in biological macromolecular crystals

Abstract

The subject matter of the present invention is an array comprising soaking solutions for soaking biological macromolecular crystals. Furthermore, the subject matter of the present invention is a rule-based method for selecting a specific soaking solution composition with a specific composition comprising a complex solute (s), water (w), a crystallization solution (crs) and / or an organic solvent (s). Furthermore, the subject matter of the present invention is a soaking solution obtained by the method of the present invention and a screening method for small molecules comprising molecular probes, fragments and drug-sized molecules using said soaking solution, and the use of said soaking solution in a screening method for small molecules on macromolecular crystals.

Description

[0001] The subject of this invention is an array of soaking solutions for soaking biological macromolecular crystals. Furthermore, the subject of this invention is a rule-based method for selecting specific soaking solution compositions having a specific composition including complex solute(s), water (w), crystallization solution (crs), and / or organic solvent(s). Additionally, the subject of this invention is the soaking solutions obtained by the method of this invention and screening methods for small molecules, including molecular probes, fragments, and drug-sized molecules, using said soaking solutions, as well as the use of said soaking solutions in methods for screening macromolecular crystals for small molecules. Background Technology

[0002] Traditional methods for identifying new drug motifs in drug discovery employ combinatorial chemistry and high-throughput screening (HTS). This has led to an ever-increasing size and complexity of chemical libraries without a corresponding increase in the number of leader structures. The vastness of the drug chemistry world seems overwhelming for any in vitro and computer-based screening method. One way to overcome this predicament is to screen small molecules called fragments. Compared to typical drug-sized compounds, fragments are smaller and have weaker binding and activity. However, the diversity and richness of information about fragments are sufficient to provide simple and straightforward compound development strategies.

[0003] Fragments need to be screened using techniques suitable for detecting inferred weak interactions. Guided by early structural affinity information determined, for example by the dissociation constant KD, higher affinity small molecules can be assembled by combining low affinity conjugates or optimizing individual low affinity conjugates.

[0004] Immersion is an important technique in crystallography-based screening, using crystals derived from biomacromolecules to generate complexes of the corresponding crystals and small molecules. According to existing techniques, biomacromolecule crystals are immersed in concentrated solutions of small molecules. Different concentrations of small molecule solutions are used depending on the molecular affinity. Higher concentrations are used for low-affinity molecules, while lower concentrations are used for high-affinity molecules. During immersion, small molecules (including molecular probes, fragments, and drug-sized molecules) diffuse into the biomacromolecule crystals and bind to the binding sites of the biomacromolecules. According to existing techniques, crystallization buffers / solutions for crystallizing biomacromolecules are extensively optimized and used as immersion solutions. Typically, such immersion solutions contain organic solvents, such as DMSO or methanol. Generally, such organic solvents, as well as small molecules and compatible solutes (penetrating agents with cryoprotective properties), have adverse effects on the crystals. It is noteworthy that crystallographic measurements are performed either at low temperatures, such as below 100 K, or at room temperature. Crystallographic measurements using diffraction techniques (X-ray diffraction, electron diffraction, neutron diffraction) at low temperatures require the use of immersion solutions with cryoprotective properties to preserve the integrity of the biological macromolecular crystal structure.

[0005] Existing technology

[0006] Traditional methods for identifying novel small molecule drug motifs in drug discovery typically employ high-throughput screening (HTS), biophysical screening cascades, phenotypic screening, and DNA-encoded library screening (DEL). This results in an ever-increasing number of small molecules in the chemical library without a corresponding increase in the number of leader structures. The vastness of the drug chemistry world seems overwhelming for any exhaustive in vitro and computational screening method. One way to overcome this predicament is to screen small molecules called fragments. Compared to typical leader or drug compounds, fragments are smaller in size and have weaker binding and activity. However, fragments are a suitable starting point for drug discovery activities because, once identified or validated crystallographically, they can be easily chemically modified and expanded based on the geometry and biophysical setting of the protein-binding pocket. Successful expansion of fragments into more drug-sized compounds yields custom compounds with desired affinity and molecular properties. Fragment methods have proven applicable even with challenging so-called “drugless” targets. Fragments require screening using techniques with appropriate sensitivity to detect the inferred weak molecular interactions. In this respect, crystallographic screening methods are unparalleled in their sensitivity to low-affinity conjugates. They can distinguish conjugates from non-conjugates with extremely low false positive and false negative rates, while elucidating the atomic analytical geometry of protein-conjugate interactions.

[0007] Crystallography-based small molecule screening involves crystallizing biomacromolecules (especially proteins), immersing the corresponding crystals of the biomacromolecules in a small molecule solution, subsequent data collection using X-ray or neutron diffraction, and finally examining the obtained dataset to identify the conjugates and elucidate the binding modes. The so-called "immersion" is a crucial technique for producing such protein-small molecule complexes. According to current techniques, experimenters typically perform this in two steps to determine the composition of the immersion solution: the immersion experiment itself and the cryopreservation of the immersion crystals.

[0008] 1. Immersion Experiment: To conduct an immersion experiment, the experimenter transfers the corresponding crystals from their original crystallization solution to an immersion solution. The immersion solution is typically based on a crystallization solution / buffer solution (or a reservoir solution if the crystals are produced using vapor diffusion), but differs from the crystallization solution due to the presence of organic solvents required to dissolve and introduce small molecules targeting biomacromolecules.

[0009] 2. Cryopreservation: For cryopreservation, the corresponding crystals are transferred from the immersion solution to a cryopreservation solution, which is typically based on a crystallization solution (or a reservoir solution if the crystals are produced using vapor diffusion), but differs from both the crystallization and immersion solutions due to the presence of cryoprotectants such as glycerol or polyethylene glycol and the lack of organic solvents. The cryoprotectant suppresses the formation of crystalline water (ice) in or around the crystals during vitrification, for example, in liquid nitrogen. Vitrification is commonly used for diffraction experiments at low temperatures (typically below 100 K) to prevent radiation damage to the crystals during high-energy X-ray measurements. Ice formation must be prevented as it can damage the crystals, and the diffraction of ice crystals can superimpose on the diffraction pattern of the protein crystals, resulting in a loss of mass in the obtained dataset. Measurements are rarely performed at room temperature. In some cases, a special cryopreservation step is not required due to the use of cryoprotectant additives during crystallization. Sometimes, crystallographers have already added a certain amount of cryoprotectant in the first step of these two steps and omit the second step. Like the other methods, this is done purely based on trial and error, without any systematic reasoning.

[0010] If suitable soaking and cryopreservation solutions are finally obtained, actual soaking experiments are conducted in the presence of a set of small molecules, such as small molecule fragments. This involves transferring protein crystals from their original crystallization environment (crystallization solution) to a soaking environment, which introduces the target small molecules dissolved in an organic solvent. In these solutions, low-affinity small molecules require higher concentrations, while high-affinity molecules require lower concentrations. To improve the hit rate of screening, experts strive to use highly concentrated small molecule solutions. Because large molecule crystals, especially proteins, are permeated by solvent channels, unlike crystals of low molecular weight compounds, small molecules can easily diffuse through crystals with sufficient solvent channel size within seconds. To improve the hit rate even for low-affinity binders, experts strive to use long soaking times (minutes, hours, days), because even low-affinity binders can be extended to high-affinity binders by chemically modifying them to target drug-like properties under the guidance of medicinal chemistry. During screening, within this immersion time, small molecules (including small molecules of different molecular weights as molecular probes, fragments, and drug-size molecules) diffuse into macromolecular crystals, especially protein crystals, and may or may not bind to the corresponding binding sites. A binding site is any location within the macromolecule that establishes a direct or solvent-mediated interaction with the corresponding small molecule. For drug-size small molecules, concentrations of 5-20 mM or higher can be used. For fragment concentrations, concentrations of 10-50 mM or higher can be used. For screening purposes, in both cases, the concentration should be as high as possible (i.e., without adverse effects on the crystals) (up to 1 M). However, as discussed below, one issue associated with adding high concentrations of small molecules dissolved in organic solvents is the effect on crystal stability.

[0011] Because biomacromolecule crystals, especially protein crystals, are highly sensitive to any changes in their environment, they often exhibit significant mass loss or even destruction when transferred from the original crystallization solution to the soaking solution. Unlike the original crystallization solution, the soaking solution contains components as organic solvents and small molecules, thus differing in composition from the original growth / crystallization solution. Since this difference adversely affects biomacromolecule crystals, successful crystallographic screening requires extensive optimization of such soaking solutions to compensate for the corresponding adverse effects on biomacromolecule crystals. According to current techniques, optimization focuses on the original crystallization solution (or, if crystals are produced via vapor diffusion), and requires extensive, unsystematic trial and error to find unique, suitable soaking solution compositions that individual crystal types can tolerate. Typically, experimenters use a plate in which soaking solutions are prepared based on the original crystallization solution (or reservoir buffer) but contain organic solvents such as DMSO for dissolving small molecule fragments. If the crystallization method is vapor diffusion, crystallologists typically use a reservoir buffer in the plate reservoir to maintain suitable osmotic conditions. In existing methods, optimization is achieved by applying all possible parameters, particularly fine-tuning the composition of the crystallization or reservoir solution used, pH adjustment, introducing additional additives (sometimes even cryoprotectants), changing the composition and volume ratio of the reservoir buffer and soaking solution, and fine-tuning the amount of organic solvent through trial and error. In particular, the presence of organic solvents can adversely affect large molecular crystals. Therefore, this results in the amount of organic solvent (typically DMSO) being usually very low, between 0.5% and 5%. The result is typically a single soaking solution composition that the crystals can tolerate. After soaking, the aforementioned cryopreservation is performed. Again, for cryopreservation purposes, the solution must be optimized to make it tolerable to the soaked crystals. This means introducing various concentration variations of cryoprotectants and other adjustments as described above.

[0012] This method can yield immersion conditions that minimize mass loss. However, since only one immersion condition is identified, it must be applied to every immersion experiment, which cannot guarantee optimal results in terms of hit rate, crystal diffraction, and other quality parameters such as mosaicism. Furthermore, in most cases, biomolecular crystals are measured at cryogenic temperatures, typically below 100 K, to prevent radiation damage. Measurements are rarely performed at room temperature. Cryogenic measurements require crystal treatment with a cryoprotectant, followed by vitrification in, for example, liquid nitrogen. Again, this cryoprotectant step has an adverse effect on the crystal, necessitating re-optimization.

[0013] The key to the method and array of this invention lies in the systematic, rule-based variation of a limited but critical parameter affecting crystal stability. The array and method of this invention can select and identify not only one soaking solution, but also a range of suitable conditions for maintaining or even improving crystal quality, and can even be used to compensate for small molecule-induced effects, such as pH changes. Another key point is that the array and method of this invention eliminates the need for two separate steps: the soaking experiment itself and cryopreservation. Furthermore, this invention introduces a systematic, rule-based method to infer suitable soaking solution composition without the need for time-consuming, unsystematic, trial-and-error-dependent optimization steps. The general components of crs, w, os, and cs are calculated using an A / B gradient algorithm to find small molecule soaking solutions equivalent to the original crystallization solution in terms of net osmotic pressure, biomacromolecule (protein) solubility, dielectric constant, and structural conformation, where os and cs are considered antagonists in terms of protein structural conformation. In fact, the array and method of this invention take into account the previously unconsidered fact that organic solvents and a class of additives called compatible solutes compensate for the adverse effects of using these two components in different steps as in the prior art. This requires balancing the corresponding concentrations of the two components, and typically also requires additional balancing with a buffer solution that maintains a certain level of ionic strength. Furthermore, this invention allows for the identification of soaking solutions with higher amounts of organic solvent, typically exceeding 5%, preferably 10%, of DMSO. Advantageously, the method of this invention also typically allows for very short or, conversely, extended soaking times of many hours (typically 6 to 48 hours), rather than the usual few seconds and minutes, thereby significantly improving the efficiency of small molecule screening of crystals. Therefore, this invention provides experts with greater possibilities and flexibility in setting up small molecule screening processes. Moreover, unlike prior art methods, the application of typically, and potentially even harmful, reservoir buffer solutions is not required.

[0014] Finally, this invention allows for the identification of novel soaking solutions aimed at reducing disorder in biomacromolecule crystals. Typically, proteins, in particular, contain disordered portions of amino acid chains that do not adopt a fixed spatial conformation. Such portions in the structure result in fuzzy electron densities that are difficult to automate data processing. The rule-based method of this invention allows for a significant increase in compatible solutes, thereby stabilizing such disordered regions. Therefore, the rule-based method using the method of this invention can significantly increase the volume of compatible solutes in a suitable soaking solution while preserving the crystal structure, maintaining the necessary regulation and balance of other essential components such as organic solvents, and can effectively screen crystals for small molecule conjugates.

[0015] Therefore, the subject matter of this invention offers experts greater possibilities and flexibility in setting up small molecule screening processes. Furthermore, unlike prior art methods, it eliminates the need for the application of reservoir buffers, which are typically, and may even be, harmful.

[0016] Finally, it is generally believed that each individual protein species of macromolecular crystal requires a separate soaking solution composition based on its original crystallization conditions. One aspect of the present invention demonstrates that this is not the case. Using the technique according to the invention, soaking solution compositions not derived from the original crystallization solution used for the nucleation, growth, and / or storage of the corresponding macromolecular crystal can be rationally selected. The inventors have demonstrated a key advantage that selected soaking solutions not derived from the original crystallization solution can be used as suitable soaking solutions for more than one type of biomacromolecular crystal. In particular, some soaking solutions of the present invention, or selected using the method of the present invention, can be used to efficiently soak crystals from different proteins. Summary of the Invention

[0017] The subject of this invention is an array for soaking biomacromolecule crystals, a method for selecting a suitable soaking solution and the composition of the soaking solution, and a method for small molecule screening using the soaking solution selected by the method of this invention.

[0018] The array of the present invention comprises a first dimension of at least two separate soaking solutions (1n to xn) and a second dimension of at least two separate soaking solutions (1m to ym), wherein each of the soaking solutions is located in a separate compartment of the array, wherein each of the soaking solutions comprises an organic solvent (os), a compatible solute (cs), a crystallization solution (crs), and water, wherein within a series of soaking solutions in the first dimension, the ratio of the volume of the compatible solute (Vcs) to the volume of the organic solvent (Vos) is the same, or, within a series of soaking solutions in the first dimension, the molar ratio of the organic solvent (os) to the compatible solute (cs) (Mos / Mcs) is the same. Within a series of soaking solutions in the first dimension, each individual soaking solution in the first dimension contains the same organic solvent and the same compatible solute, wherein the ratio of water volume Vw to crystallization solution volume Vcrs varies in the series of individual soaking solutions in the first dimension, wherein one of the series of individual soaking solutions in the first dimension has a minimum or zero Vcrs and a maximum Vw, and another individual soaking solution in the first dimension has a minimum or zero Vw and a maximum Vcrs, wherein the other individual soaking solutions in the first dimension take Vcrs and Vw values ​​between the two aforementioned values.

[0019] In one specific embodiment of the invention, 1n to xn defines the number of individual soaking solutions in the range of 1 (1n) to 1×106 (xn), wherein the number includes, but is not limited to, 1×106 or 1×105, 1×104 or 768 or 384 or 96 or 48 or 24, however, the arrangement of the array determines the number of individual soaking solutions.

[0020] In another specific embodiment of the invention, 1m to ym defines the number of individual soaking solutions in the range of 1 (1m) to 1×106 (ym), wherein the number includes, but is not limited to, 1×106 or 1×105, 1×104 or 768 or 384 or 96 or 48 or 24, however, the arrangement of the array determines the number of individual soaking solutions.

[0021] The subject of this invention is an array according to the invention, wherein the array comprises a second dimension (dII) of a series of individual soaking solutions of biomacromolecule crystals, wherein the compatibility solute varies in the second dimension and depends on the compatibility solute used in the series of soaking solutions in the second dimension, wherein the ratio of the volume of the compatibility solute (Vcs) to the volume of the organic solvent (Vos) or the molar ratio of the organic solvent (os) to the compatibility solute (cs) (Mos / Mcs) may be different.

[0022] In one embodiment, in each of the series of said individual soaking solutions, the ratio of the volume of the compatible solute Vcs to the volume of the organic solvent Vos is the same, said volume ratio being from 1:1,000,000 to 1,000,000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos).

[0023] In one embodiment, in each of the series of said individual soaking solutions, the molar ratio of organic solvent (os) to compatible solute (cs) is the same (Mos / Mcs), said molar ratio (Mos / Mcs) being 1:10000 to 10000:1, preferably between 100:1 and 1:100, more preferably between 10:1 and 1:10.

[0024] The subject of this invention is an array according to the invention, wherein the array comprises at least x series in a first dimension (1n to xn) and a second dimension (1m to ym) of individual soaking solutions for soaking biomacromolecule crystals, wherein the compatible solutes or other components of the soaking solutions in the x series of individual soaking solutions in the second dimension can vary. Variations in dimension dII may be due to the number of compatible solutes tested in the array, or variations in dimension dII may be due to the number of combinations of compatible solutes and / or organic solvents tested in the array.

[0025] The subject of this invention is an array according to the invention, wherein the total volume of each individual soaking solution is typically in the range of μm to ml, typically between 2.5 μl and 10 μl.

[0026] Unless the volume is explicitly expressed in a standard volume unit such as μm or ml, "V" (as in Vos, Vcs, Vcrs, and Vw) always refers to the proportion / percentage of the relevant component in the total volume (VT) of the solution, i.e., 100%. For example, Vos refers to the percentage / percentage of organic solvent (os) in the total volume of the solution; for example, Vos=10 means that os accounts for 10% of the total volume of the solution.

[0027] The molar ratio Mos / Mcs refers to the ratio of the number of moles of organic solvent (os) to the number of moles of compatible solute (cs).

[0028] The ratio of organic solvent (os) to liquid compatible solute (cs) can be defined as the ratio of the volume of compatible solute Vcs to the volume of organic solvent Vos, i.e., Vcs:Vos, or as the molar ratio of organic solvent (os) to compatible solute (cs) Mos / Mcs. These two definitions are considered equivalent within the scope of this invention. The ratio of organic solvent (os) to solid compatible solute (cs) is defined by the molar ratio of organic solvent (os) to compatible solute (cs) Mos / Mcs. The effect provided by mixing liquid compatible solute (cs) and organic solvent (os) in a specified molar ratio is the same as mixing solid compatible solute (cs) and organic solvent (os) in the same molar ratio.

[0029] As mentioned earlier, the ratio of liquid compatible solute (cs) to organic solvent (os) can be defined as the ratio of the volume of compatible solute Vcs to the volume of organic solvent Vos, i.e., Vcs:Vos, or it can be defined as the concentration Ccs (mol / L) or the number of moles of compatible solute Ncs / volume of organic solvent Vos, i.e., Ccs=Ncs:Vcs. These two definitions are considered equivalent within the scope of this invention. The ratio of solid compatible solute (cs) to organic solvent (os) can be defined as the concentration Ccs or the number of moles of compatible solute Ncs / volume of organic solvent Vos, respectively.

[0030] Ncs refers to the number of moles of the compatible solute (cs), and Nos refers to the number of moles of the organic solvent (os).

[0031] Depending on the required precision in the specified array, the preparation of the solution (e.g., by pipetting techniques) may vary. If high precision is required, low-volume pipetting in the picoliter to microliter range can be performed using automated pipetting (i.e., pipetting robots). However, for logical reasons, volumes in the microliter to milliliter range are preferred for the array, as they can be manually pipetted (i.e., with technical assistance). However, the volume that can be used in the array is limited only by the plate used for soaking (either custom-made in, for example, a 3D printer or commercially available).

[0032] In one specific embodiment of the invention, the soaking time ranges from a few seconds to 24 months, preferably up to 52 weeks, and more preferably 72 hours. Preferably, the soaking time is 6 hours to 24 hours.

[0033] The subject of this invention is an array according to the invention, wherein the compatible solute is selected from polyols, amino acids, methylamine and other compatible solutes.

[0034] In one specific embodiment of the invention, the array comprises a compatible solute, wherein the polyhydroxy compound is selected from polyoxyethylene, polyols, monosaccharides, disaccharides, trisaccharides, sugar alcohols, cyclic polyols, and derivatives of the aforementioned molecules. Mixtures of any of the compatible solutes may also be used.

[0035] In another specific embodiment of the present invention, the polyoxyethylene and polyol group comprises monodisperse or polydisperse polyoxyethylene with a molecular weight in the range of 20-2,000,000 g / mol, including but not limited to, for example, polyethylene glycol 400, and polyols including propylene-1,2,3-triol (glycerol), ethylene-1,2-diol (ethylene glycol), 2-methylpentan-2,4-diol (2-methylpentan-2,4-diol), and (3R,4S,5S,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxacyclohexane-3,4,5-triol (1-glucosylglycerol).

[0036] In another specific embodiment of the present invention, the monosaccharide group preferably includes, but is not limited to, for example, (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (glucose), (3S,4R,5R)-1,3,4,5,6-pentahydroxyhexan-2-one (fructose), and (2S,3R,4S,5R,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxacyclohexyl-3,4,5-triol (isofloridoglycoside).

[0037] In another specific embodiment of the present invention, the disaccharide group preferably includes, but is not limited to, for example, (β-D-fructofuranosyl-α-D-glucopyranoside (sucrose) and (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxecyclohex-2-yl]oxyoxecyclohex-3,4,5-triol (trehalose).

[0038] In another specific embodiment of the invention, the trisaccharide group preferably includes, but is not limited to, for example, (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxetane-2-yl]oxo-6-[[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxetane-2-yl]oxomethyl]oxetane-3,4,5-triol (raffinose).

[0039] In another specific embodiment of the present invention, the sugar alcohol group preferably includes, but is not limited to, for example, (2R,3R,4R,5R)-hexane-1,2,3,4,5,6-hexane (mannitol), (2S,3R,4R,5R)-hexane-1,2,3,4,5,6-hexane (sorbitol), (1R,2S,3r,4R,5S,6s)-cyclohexane-1,2,3,4,5,6-hexane (inositol), (2R,3 (R,4S)-pentan-1,2,3,4,5-pentanol (xylitol), (2R,3s,4S)-pentan-1,2,3,4,5-pentanol (arabinitol), (2R,3S)-butan-1,2,3,4-tetraol (erythritol), (2R,4R)-pentan-1,2,3,4,5-pentanol (arabinitol), and (2R,3S,4R,5S)-hexan-1,2,3,4,5,6-hexaneol (galactitol).

[0040] In another embodiment of the invention, the cyclic polyol group preferably includes, but is not limited to, for example, (1S,2S,4S,5R)-6-methoxycyclohexyl-1,2,3,4,5-pentaol (pine alcohol) and (2S,3R,4S,5R,6R)-2-[(1,3-dihydroxypropyl-2-yl)oxy]-6-(hydroxymethyl)oxacyclohexyl-3,4,5-triol (floridoside).

[0041] In an additional specific embodiment of the invention, the group of derivatives of the aforementioned molecules preferably includes, but is not limited to, for example, mannose derivatives such as fiorin ((2R)-O2-(β-D-mannopyranosyl)glyceric acid) and fiorin-A (mannosylglyceramide), inositol derivatives (preferably diinositol-1,1'-phosphate), glycerol derivatives (preferably cyclic 2,3-diphosphoglycerate, α-diglycerol phosphate), and polymers of the aforementioned molecules, such as starch, fructan, cellulose (all possible stereoisomers).

[0042] In another specific embodiment of the invention, the array comprises a compatible solute, wherein the polyhydroxy compound is selected from the preferred group comprising: polyoxyethylene with a molecular weight in the range of 200-20000 g / mol, including but not limited to polyoxyethylene (200 g / mol, 300 g / mol, 400 g / mol, 550 g / mol, 600 g / mol, 1000 g / mol, 1500 g / mol, 2000 g / mol, 3350 g / mol, 4000 g / mol, 5000 g / mol, 6000 g / mol, 8000 g / mol, 10000 g / mol, 20000 g / mol). (g / mol), selected from propan-1,2,3-triol (glycerol), ethylene-1,2-diol (ethylene glycol), 2-methylpentan-2,4-diol (2-methylpentan-2,4-diol), and polyols selected from (3S,4R,5R)-1,3,4,5,6-pentahydroxyhexan-2-one (fructose), (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (glucose), (2S,3R,4R,5R)-hexan-1, Sugars of 2,3,4,5,6-hexanediol (sorbitol), (β-D-fructofuranosyl-α-D-glucopyranoside (sucrose), and sugars of (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxecyclohex-2-yl]oxyoxecyclohex-3,4,5-triol (trehalose), and inositol derivatives (preferably diinositol-1,1'-phosphate).

[0043] In another specific embodiment of the invention, the array comprises a compatible solute, wherein the polyhydroxy compound is selected from the more preferred group including: polyoxyethylene with a molecular weight in the range of 400-20000 g / mol, including but not limited to polyoxyethylene (400 g / mol, 550 g / mol, 3350 g / mol, 6000 g / mol, 8000 g / mol, 10000 g / mol, 20000 g / mol). (g / mol) polyols selected from propan-1,2,3-triol (glycerol), ethylene-1,2-diol (ethylene glycol), 2-methylpentan-2,4-diol (2-methylpentan-2,4-diol), and sugars selected from β-D-fructofuranosyl-α-D-glucopyranoside (sucrose), (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (glucose), (2S,3R,4R,5R)-hexyl-1,2,3,4,5,6-hexane (sorbitol), and (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxecyclohexyl-2-yl]oxyoxecyclohexyl-3,4,5-triol (trehalose).

[0044] In an additional embodiment of the invention, the array comprises a compatible solute, wherein the polyhydroxy compound is selected from the most preferred group comprising: polyoxyethylene with a molecular weight in the range of 400-20000 g / mol, including but not limited to polyoxyethylene (400 g / mol). (g / mol) , selected from polyols such as propan-1,2,3-triol (glycerol), ethylene-1,2-diol (ethylene glycol), and 2-methylpentan-2,4-diol (2-methylpentan-2,4-diol), or sugars selected from (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxecyclohexyl-2-yl]oxyoxecyclohexyl-3,4,5-triol (trehalose), (2S,3R,4R,5R)-hexyl-1,2,3,4,5,6-hexane (sorbitol), and β-D-fructofuranosyl-α-D-glucopyranoside (sucrose). Mixtures of compatible solutes such as polyoxyethylene may also be used.

[0045] According to the present invention, the amino acid group (with the amino group in the α and β positions relative to the formate group) preferably includes 2-aminoethanesulfonic acid (taurine), 2-aminoethanesulfinic acid (hypotaurine), (2S)-pyrrolidine-2-carboxylic acid (proline), 2-aminoacetic acid (glycine), (6S)-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (ectoine), (5S,6S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (hydroxyectoine), 4-aminobutyric acid (γ-aminobutyric acid), ( 2S)-2-aminoglutaric acid (glutamic acid), β-hydroxy-γ-N-trimethylaminobutyric acid, (2S)-2-amino-3-methylbutyric acid (valine), (2S,3S)-2-amino-3-methylvaleric acid (isoleucine), (2S)-2-aminosuccinic acid (aspartic acid), (2S)-2-aminopropionic acid (alanine), 2-(methylamino)ethanesulfonic acid (N-methyltaurine), (2S)-2-[[(1R)-1-carboxyethyl]amino]-5-(diaminomethyleneamino)valeric acid (Octopin). It should be noted that the amino acids selected from the preferred group can appear in all possible stereoisomers. Mixtures of any of the above-mentioned compatible solutes can also be used.

[0046] According to the present invention, the methylamine group preferably includes N,N-dimethylmethylamine oxide (trimethylamine N-oxide or TMAO), 2-trimethylammonium acetate (trimethylglycine or betaine), 2-(methylamino)acetic acid (N-methylglycine or sarcosine), 2-hydroxyethyl(trimethyl)ammonium (choline), 2-[formamidinyl(methyl)amino]acetic acid (creatine), L-α-glycerophosphorylcholine, and (R)-(3-carboxy-2-hydroxypropyl)-N,N,N-trimethylammonium hydroxide (carnitine).

[0047] According to the present invention, other compatible solute groups include: 1-methyl-2-pyridinecarboxylic acid (lobster muscle), methyl sulfonate solutes such as 3-dimethylsulfonylpropyl ester (dimethylsulfonyl propyl ester), and methyl sulfoxides such as dimethyl sulfoxide, urea (and derivatives), and other compounds that, by definition, have the properties of compatible solutes.

[0048] The subject of this invention is an array according to the invention, wherein the compatible solute is selected from the most preferred group including PEG400, MPD, EG and glycerol.

[0049] According to the present invention, mixtures of single compatible solutes selected from different groups and the same group, including polyhydroxy compounds, amino acids, methylamine, and other compatible solutes, can be used in the array. Therefore, the term "compatible solute" according to the present invention can refer to a mixture of compatible solutes. In a preferred embodiment, cs is a PEG mixture, more preferably PEG400 and PEG3350.

[0050] In a preferred embodiment of the present invention, a method for selecting the composition of an immersion solution is provided, wherein the compatible solute is selected from polyhydroxy compounds, amino acids, methylamine, or mixtures thereof, wherein the polyhydroxy compounds are selected from polyoxyethylene, polyols, monosaccharides, disaccharides, trisaccharides, sugar alcohols, cyclic polyols, and derivatives of the aforementioned molecules, wherein the amino acids are selected from 2-aminoethanesulfonic acid (taurine), 2-aminoethanesulfinic acid (hypoturic acid), (2S)-pyrrolidine-2-carboxylic acid (proline), 2-aminoacetic acid (glycine), (6S)-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (ectoin), (5S,6S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (hydroxyectoin), 4-aminobutyric acid (γ-aminobutyric acid), (2S)-2-aminoglutamate (glutamic acid), β-hydroxy-γ-N-trimethylaminobutyric acid, (2S)-2- Amino-3-methylbutyric acid (valine), (2S,3S)-2-amino-3-methylvaleric acid (isoleucine), (2S)-2-aminosuccinic acid (aspartic acid), (2S)-2-aminopropionic acid (alanine), 2-(methylamino)ethanesulfonic acid (N-methyltaurine), (2S)-2-[[(1R)-1-carboxyethyl]amino]-5-(diaminomethyleneamino)valeric acid (Octopin), wherein the methylamine is selected from N N-Dimethylamine oxide (trimethylamine N-oxide or TMAO), 2-Trimethylammonium acetate (trimethylglycine or betaine), 2-(methylamino)acetic acid (N-methylglycine or sarcosine), 2-hydroxyethyl(trimethyl)ammonium (choline), 2-[formamidinyl(methyl)amino]acetic acid (creatine), L-α-glycerophosphorylcholine, (R)-(3-carboxy-2-hydroxypropyl)-N,N,N-trimethylammonium hydroxide (carnitine).

[0051] In another preferred embodiment of the invention, a method for selecting the composition of an immersion solution is provided, wherein the compatible solute is selected from polyhydroxy compounds, amino acids, methylamine, or mixtures thereof, wherein the polyhydroxy compounds are selected from polyoxyethylene, polyols, monosaccharides, disaccharides, trisaccharides, sugar alcohols, cyclic polyols, and derivatives of the aforementioned molecules, wherein the polyoxyethylene group is selected from molecules with a molecular weight between 20 and 2,000,000. Monodisperse or polydisperse polyethylene oxides in the range of g / mol, including but not limited to, for example, polyethylene glycol 400 and polyethylene glycol 600, wherein the polyol is selected from propylene-1,2,3-triol (glycerol), ethylene-1,2-diol (ethylene glycol), 2-methylpentan-2,4-diol (2-methylpentan-2,4-diol), and (3R,4S,5S,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxacyclohexane-3,4,5-triol (1-glucosylglycerol), wherein the monosaccharide is selected from (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanol (glucosylglycerol). (glucose), (3S,4R,5R)-1,3,4,5,6-pentahydroxyhexane-2-one (fructose), (2S,3R,4S,5R,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxacyclohexyl-3,4,5-triol (isofloridoside), wherein the disaccharide group is selected from β-D-fructofuranosyl α-D-glucopyranoside (sucrose), (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxacyclohexyl-2-yl]oxyoxacyclohexyl-3,4 ,5-Triol (trehalose), wherein the trisaccharide group is selected from (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxetane-2-yl]oxo-6-[[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxetane-2-yl]oxomethyl]oxetane-3,4,5-triol (raffinose), wherein the sugar alcohol group includes (2R,3R,4R,5R)-hexyl-1,2,3,4,5,6-hexane (mannitol), (2S,3R,4R,5R)-hexyl-1,2,3,4,5,6-hexane (mannitol), (2S,3R,4R,5R)-hexane ... The following are listed: (1R,2S,3r,4R,5S,6s)-cyclohexyl-1,2,3,4,5,6-hexyl alcohol (sorbitol), (2R,3R,4S)-pentan-1,2,3,4,5-pentanol (xylitol), (2R,3s,4S)-pentan-1,2,3,4,5-pentanol (ardentitol), (2R,3S)-butan-1,2,3,4-tetraol (erythritol), (2R,4R)-pentan-1,2,3,4,5-pentanol (arabinitol), and (2R,3S,4R,5S)-hexyl-1,2,3,4,5,6-hexyl alcohol (sorbitol).6-Hexahexol (galactitol), wherein the cyclic polyol group includes (1S,2S,4S,5R)-6-methoxycyclohexyl-1,2,3,4,5-pentaol (pineol) and (2S,3R,4S,5R,6R)-2-[(1,3-dihydroxypropyl-2-yl)oxy]-6-(hydroxymethyl)oxacyclohexyl-3,4,5-triol (floridoside), wherein the derivative group of the aforementioned molecules includes mannose derivatives such as fiorin ((2R)-O2-(β-D-mannopyranosyl)glyceric acid) and fiorin-A (mannosylglyceramide), inositol derivatives (preferably diinositol-1,1'-phosphate), glycerol derivatives (preferably cyclic 2,3-diphosphoglycerate, α-diglyceride), and polymers of the aforementioned molecules, such as starch, fructan, and cellulose.

[0052] In one embodiment of the array described above, the array includes a first dimension comprising a series of at least two separate soaking solutions for soaking biomolecules, wherein the compatible solute in the second dimension of the series of at least two separate soaking solutions differs from the compatible solute in the first dimension. In another embodiment of the array, the array includes 1x to xn series of separate soaking solutions for biomolecules in the first dimension, wherein the compatible solute in each of the separate soaking solution series in the first dimension differs from the compatible solute in the separate soaking solution series in the second dimension.

[0053] The subject of this invention is an array according to the invention, wherein the organic solvent comprises low-reactive proton or aprotic liquid carbohydrates capable of dissolving small molecules. Examples of organic solvents include, but are not limited to: straight-chain or branched monoaliphatic alcohols, such as methanol, ethanol, propan-1-ol, propan-2-ol, 2-methylpropan-1-ol (isobutanol), butan-1-ol (n-butanol), and 2-methylpropan-2-ol (tert-butanol); di- or poly-alliphatic alcohols, such as 2-methyl-2,4-pentanediol, hexane-2,5-diol (2,5-hexanediol), propan-1,3-diol (1,3-propanediol), butan-2,3-diol, propan-1,2-diol (1,2-propanediol), and ethylene-1,2-diol (ethylene glycol); monoalkyl ethers of aliphatic diols, such as monomethyl ethers of ethylene glycol; dialkyl ketones, such as propan-2-one (acetone); cyclic non-aromatic ethers, such as oxapentane (tetrahydrofuran), crown ethers, and 1,4-dialkyl ethers. Alkane (di) Alkane); cycloalkenyl ethers, such as furan; benzyl alcohol; phenethyl alcohol; (alkylated) sulfoxides, such as methyl sulfoxide methane (DMSO) and its derivatives; esters derived from acetic acid, such as ethyl acetate and its derivatives; alkylated formamides, such as N,N-dimethylformamide; organic nitriles, such as acetonitrile; lactones, such as oxepane-2-one (γ-butyrolactone), 4-methyloxepane-2-one (β-butyrolactone); polyethers, such as polyethylene glycol (e.g., 400); polyetheramines, such as Jeffamine (e.g., 400); sugar ethers, such as 2-(2-ethoxyethoxy)ethanol (diethylene glycol); and other organic solvents suitable for dissolving small molecules for protein crystal-based screening.

[0054] In one embodiment of the invention, there is a method for selecting the composition of an immersion solution, wherein the organic solvent is a straight-chain or branched monoaliphatic alcohol, a dialiphatic alcohol, a monoalkyl ether of an aliphatic diol, an ether of aliphatic monoaliphatic alcohol, a cyclic non-aromatic ether, a (cyclic)enol ether, a dialkyl ketone, benzyl alcohol, phenethyl alcohol, (alkylated) sulfoxide and corresponding phosphorus derivatives, an ester derived from acetic acid, (alkylated) formamide or a mixture thereof.

[0055] In one aspect, the present invention is an array according to the invention, wherein the crystallization solution crs obtained from the crystallization process of the said biomacromolecule crystals comprises pH and buffering substances, additives, and precipitants. According to the invention, mixtures of individual components of a crystallization solution selected from different groups and the same group comprising individual components of a crystallization solution including pH and buffering substances, additives, and precipitants can be used in the array, soaking solution, and method of the invention.

[0056] According to the present invention, pH and buffering substances typically include acetate buffer, TRIS buffer, HEPES buffer, dimethyl arsenate buffer, 1H-imidazole (imidazole) buffer, citrate buffer, and others.

[0057] According to the present invention, precipitants typically include volatile organic solvents, salts, polymers, and non-volatile organic solvents.

[0058] In one specific embodiment of the invention, the preferred volatile organic solvent group typically includes ethanol, propan-1-ol, propan-2-ol, 2-propanol, and 1,4-diol. Alkane (di) Alkane), prop-2-one (acetone), 2-methylprop-1-ol (isobutanol), but-1-ol (n-butanol), 2-methylprop-2-ol (tert-butanol), acetonitrile, methyl sulfoxide (dimethyl sulfoxide), oxepane-2-one (γ-butyrolactone), 4-methyloxepane-2-one (β-butyrolactone).

[0059] In another specific embodiment of the invention, the preferred salt group typically includes disodium 2,3-dihydroxysuccinate (sodium tartrate), (2R,3R)-2,3-dihydroxysuccinate (dipotassium tartrate), ammonium phosphate (ammonium phosphate), diammonium sulfate (ammonium sulfate), triammonium 2-hydroxypropyl-1,2,3-tricarboxylate (ammonium citrate), sodium acetate (sodium acetate), ammonium acetate (ammonium acetate), dilithium sulfate, lithium chloride, lithium acetate, lithium formate, lithium nitrate, sodium nitrate, sodium chloride, potassium chloride, sodium formate, sodium and potassium monophosphates, and sodium and potassium diphosphates. Salts and polyphosphates, sodium citrate (such as sodium 2-hydroxypropyl-1,2,3-tricarboxylate dihydrogen phosphate (mono-sodium citrate), disodium 2-hydroxypropyl-1,2,3-tricarboxylate hydrogen phosphate (disodium citrate), trisodium 2-hydroxypropyl-1,2,3-tricarboxylate (trisodium citrate), magnesium dicarboxylate, magnesium dichloride, magnesium sulfate, calcium dichloride dihydrate, disodium succinate (sodium succinate), cadmium(II) sulfate (cadmium sulfate), disodium malonate (sodium malonate), magnesium diacetate (magnesium acetate), zinc diacetate (zinc acetate), calcium diacetate (calcium acetate).

[0060] In another specific embodiment of the invention, the preferred polymer group typically includes polyethylene glycol (preferably PEG400, PEG1000, PEG3350, PEG4000, PEG6000, PEG8000, PEG20000), Jeffamine T, Jeffamine M, 2-methoxyethanol (polyethylene glycol monomethyl ester), 2-hydroxyethyl octadecanoate (polyethylene glycol monostearate), and polyenamine. PEG can be used in pure or diluted form, for example, diluted 50% in water.

[0061] In an additional specific embodiment of the invention, the preferred group of non-volatile organic solvents includes 2-methyl-2,4-pentanediol, hexane-2,5-diol (2,5-hexanediol), propylene-1,3-diol (1,3-propanediol), polyethylene glycol (e.g., 400), and Jeffamine (e.g., 400).

[0062] According to the present invention, the additive group includes physiologically or biochemically relevant small molecules, chemical protectants, solubilizers and detergents, compounds that reduce twinning, penetrants, cosolvents and structure-making agents, categories of compounds that crosslink carboxyl and amino groups on protein surfaces, categories of compounds that form electrostatic, covalent, or hydrogen-bonded arrangements aimed at stabilizing biomacromolecule crystals, and categories of substances or compounds used to enhance nucleation and provide unique surfaces.

[0063] In one specific embodiment of the invention, with regard to proteins, the preferred additive group includes physiologically or biochemically relevant small molecules, such as 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiophene[3,4-d]imidazol-4-yl]valerate biotin, [[(2R,3S,4R,5R)-5-(6-aminopurine-9-yl)-3,4-dihydroxyoxacyclopentan-2-yl]methoxy-hydroxyphosphoryl]phosphonate hydrogen phosphate (adenosine triphosphate), [(2R,3S,4R,5R)-5-(6-aminopurine-9-yl)-3,4-dihydroxyoxacyclopentan-2-yl]methoxy-hydroxyphosphoryl]phosphonate hydrogen phosphate (adenosine triphosphate), [(2R,3S,4R, 5R)-5-(6-aminopurine-9-yl)-3,4-dihydroxyoxacyclopentan-2-yl]methylphosphonic acid hydrogen phosphate (adenosine diphosphate), [(2R,3S,4R,5R)-5-(6-aminopurine-9-yl)-3,4-dihydroxyoxacyclopentan-2-yl]methyl dihydrogen phosphate (adenosine monophosphate), 3-[18-(2-carboxyethyl)-7,12-bis(vinyl)-3,8,13,17-tetramethyl-21H,23H-porphyrin-2-yl]ferric propionate (Fe-protoporphyrin IX or heme b).

[0064] In another specific embodiment of the invention, the preferred additive group includes chemical protective agents, such as reducing agents, like 2-mercaptoethanol (β-mercaptoethanol or BME) and (2S,3S)-1,4-bis(mercapto)but-2,3-diol (dithiothreitol or DTT), 3-[bis(2-carboxyethyl)phosphono]propionate (TCEP), heavy metal ion scavengers such as 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid (ethylenediaminetetraacetic acid or EDTA) and 2-[2-[2-[2-[2-[bis(carboxymethyl)amino]ethoxy]ethoxy]ethyl-(carboxymethyl)amino]acetic acid (EGTA), compounds that prevent microbial infection such as sodium azide (sodium azide), phenol, and 1,1,1-trichloro-2-methylprop-2-ol (chlorobutanol).

[0065] In another specific embodiment of the invention, the preferred additive group includes solubilizers and detergents, such as quaternary ammonium salts; sulfobetaine; dissociating agents, such as urea; and surfactants and detergent molecules, such as (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-octaoxyoxacyclohexane-3,4,5-triol (n-octyl-β-d-glucoside), octyl-polyoxyethylene (octyl-POE), N,N-dimethyldodecane-1-amine oxide (lauryl dimethylamine oxide), (2R,3R... ,4S,5S,6R)-2-[(2R,3S,4R,5R,6R)-6-dodecyloxy-4,5-dihydroxy-2-(hydroxymethyl)oxacyclohexyl-3-yl]oxo-6-(hydroxymethyl)oxacyclohexyl-3,4,5-triol (dodecyl-β-D-maltodextrin), sodium 1,4-bis(2-ethylhexyloxy)-1,4-dioxobut-2-sulfonate (bis(2-ethylhexyl)sulfosuccinate), dodecyl ammonium chloride (dodecyl ammonium chloride), polyethoxylated fatty acids.

[0066] In an additional specific embodiment of the invention, the preferred additive group includes compounds intended to reduce twinning, such as ethanol, methyl sulfoxide (dimethyl sulfoxide or DMSO), acetone (acetone), and 1,4-dimethyl sulfoxide (DMSO). Alkane (di) Alkane), but-1-ol (butanol), 2-methylpentane-2,4-diol (2-methyl-2,4-pentanediol or MPD).

[0067] In another specific embodiment of the invention, the preferred additive group includes a penetrant, a co-solvent, and a structure-forming agent, such as (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxetane-2-yl]oxo-6-(hydroxymethyl)oxetane-3,4,5-triol (sucrose), (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3, 4,5-Trihydroxy-6-(hydroxymethyl)oxetine-2-yl]oxyoxetine-3,4,5-triol (trehalose) and other sugars, (2S)-pyrrolidine-2-carboxylic acid (proline), N,N-dimethylamine oxide (trimethylamine N-oxide or TMAO), 2-aminoacetic acid (glycine), 2-(trimethylammonium)acetate (betaine), 2-aminoethanesulfonic acid (taurine), 2-(methylamino)acetic acid (sarcosine), and others and their corresponding stereoisomers (if present).

[0068] In another specific embodiment of the invention, the preferred additive group includes a class of compounds that crosslink carboxyl and amino groups on the protein surface, such as molecules containing diamino or dicarboxylic acids or aliphatic moieties of varying lengths carrying charged groups.

[0069] In another specific embodiment of the invention, the preferred additive group includes a class of compounds that form electrostatic, covalent, or hydrogen-bonded arrangements of crystals by means of intermolecular crosslinking between proteins in the crystal, such as glutaraldehyde.

[0070] In another specific embodiment of the invention, the preferred additive group includes categories of substances or compounds used to enhance nucleation and provide unique surfaces, such as PEG, Jeffamine, gels, such as gels used for cubic lipid phase crystallization, and surfaces that promote epitaxial and heterogeneous nucleation, commercially available nucleating agents such as Crystallophore No. 1, Naomi's nucleating agent, JBS Agical Triangle, Anderson-Evans polyoxotungstate (Na6[TeW6O24]·22H2O), or buffer solution components that affect the solubility and aggregation properties of macromolecules and thus affect their nucleation and growth properties, such as 2-aminoacetic acid (glycine), 3-[4-(3-sulfopropyl)piperazin-1-yl]prop-1-sulfonic acid (PIPPS), 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)prop-1,3-diol (Bis-Tris), 2-morpholino-4-ylethanesulfonic acid (MES), 2-[(2-amino-2-oxoethyl) [1,3-(carboxymethyl)amino]acetic acid (ADA), 2-[3-[[1,3-dihydroxy-2-(hydroxymethyl)prop-2-yl]amino]propylamino]-2-(hydroxymethyl)prop-1,3-diol (Bis-Tris propane), 3-morpholino-4-ylprop-1-sulfonic acid (MOPS), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-amino-2-(hydroxymethyl)prop-1,3-diol (TRIS) ), 3-[4-(2-hydroxyethyl)piperazin-1-yl]prop-1-sulfonic acid (EPPS), 1H-imidazolium (imidazolium), 2-[bis(2-hydroxyethyl)amino]acetic acid (dihydroxyethylglycine), 2-(cyclohexylamino)ethanesulfonic acid (CHES), 3-(cyclohexylamino)prop-1-sulfonic acid (CAPS), (2R,3S,4S,5R)-2-(hydroxymethyl)-6-octaoxyoxacyclohexyl-3,4,5-triol (octylpyranose glucoside) (2R,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6R)-6-dodecyloxy-4,5-dihydroxy-2-(hydroxymethyl)oxacyclohex-3-yl]oxo-6-(hydroxymethyl)oxacyclohex-3,4,5-triol (dodecyl maltoside), (2S,3S)-1,4-bis(mercapto)but-2,3-diol (DTT), and 3-[bis(2-carboxyethyl)phosphono]propionic acid (TCEP).

[0071] Cryoprotectants include polyethylene glycols such as PEG200, PEG400, P600, etc.; ethylene-1,2-diol (ethylene glycol); 2-methylpentane-2,4-diol (2-methyl-2,4-pentanediol or MPD); N,N-dimethylamine oxide (trimethylamine N-oxide or TMAO); 2-trimethylammonium acetate (trimethylglycine or betaine); 2-(methylamino)acetic acid (N-methylglycine or sarcosine); propionyl-1,2,3-triol (glycerol); (2S,4R... (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxetane-2-yl]oxo-6-(hydroxymethyl)oxetane-3,4,5-triol (sucrose), (3R,4S,5S,6R)-6-(hydroxymethyl)oxetane-2,3,4,5-tetraol (Glucose), (2S,3R)-Butyl-1,2,3,4-tetraol (erythritol), propan-1-ol (propanol), propan-2-ol (isopropanol), ethanol, 2-ethoxyethanol (ethylene glycol monoethyl ether), (2R,3R)-Butan-2,3-diol; (1R,2R)-cyclohexyl-1,2-diamine ((2R,3R)-Butan-2,3-diol), (2R,3S,4S,5R,6R)-2-(hydroxymethyl) (2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxacyclohexyl-2-yl]oxyoxacyclohexyl-3,4,5-triol (trehalose), 2-aminoethanesulfonic acid (taurine), (6S)-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (ectoine), (2S)-pyrrolidine-2-carboxylic acid (proline), and others, as well as derivatives and stereoisomers.

[0072] In this application, crystallization solution (crs) refers to both 1) the crystallization solution crs obtained from the crystallization process of biological macromolecule crystals described above, and 2) newly prepared solutions of chloride salts such as NaCl, MgCl2 and KCl.

[0073] In the prior art, it is assumed that each individual protein species requires a separate soaking solution composition based on its original crystallization conditions for its macromolecular crystals. One aspect of the present invention demonstrates that this is not the case. Using the technique according to the invention, it is reasonable to screen soaking solution compositions that are not derived from the original crystallization solutions used for the nucleation, growth, and / or storage of the corresponding macromolecular crystals. Conversely, contrary to intuition, salt solutions such as NaCl or KCl, or any other salt generally suitable for crystallization experiments or for use in buffer compositions, can maintain the integrity of macromolecular crystals. The method according to the invention allows for the identification of suitable combinations and concentrations of salt types, compatible solutes, and organic solvents. This demonstrates that soaking solutions can be independent of the original crystallization conditions. Interestingly, contrary to intuition, very different crystal species grown under very different crystallization conditions can be stabilized and soaked in several soaking solutions identified by applying one embodiment of the invention.

[0074] The newly prepared CRS consists of a salt solution that is not the original CRS but is equivalent in terms of net osmotic pressure, biomolecular solubility, dielectric constant, and structural conformation. It maintains a certain level of ionic strength or buffering capacity and preserves crystal integrity. Typically, the newly prepared CRS is referred to as an equivalent salt solution different from the original CRS used for the nucleation, growth, and / or storage of macromolecular crystals, where the salt in the solution is any salt suitable for the crystallization of biomolecular molecules.

[0075] Another embodiment of the invention is a method for selecting the composition of an immersion solution, wherein an equivalent salt solution different from the crystallization solution is used, wherein the salt is selected from disodium 2,3-dihydroxysuccinate (sodium tartrate), (2R,3R)-2,3-dihydroxysuccinate (dipotassium tartrate), ammonium phosphate (ammonium phosphate), diammonium sulfate (ammonium sulfate), triammonium 2-hydroxypropyl-1,2,3-tricarboxylate (ammonium citrate), sodium acetate, ammonium acetate, dilithium sulfate, lithium chloride, lithium acetate, lithium formate, lithium nitrate, sodium nitrate, sodium chloride, potassium chloride, sodium formate, monophosphates of sodium and potassium, diphosphates and polyphosphates of sodium and potassium, sodium citrate (such as sodium dihydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (monosodium citrate), disodium hydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (disodium citrate)). The equivalent solution contains salts preferably selected from lithium acetate, lithium chloride, lithium formate, lithium nitrate, dilithium sulfate, magnesium dimethyl sulfide, magnesium dimethyl sulfide, magnesium dimethyl sulfide, magnesium dimethyl sulfide, magnesium dimethyl sulfide, magnesium diacetate, zinc diacetate, and calcium diacetate.

[0076] In another embodiment of the invention, the equivalent aqueous solution is suitable for storing macromolecular crystals and replacing the original CRS, while helping to maintain crystal integrity and preserve parameters, particularly in terms of net osmotic pressure, dielectric constant, solubility of the corresponding biomacromolecules, and influence on the structural conformation of the biomacromolecules. It is not intended to be unsuitable for promoting protein nucleation and crystal growth or for storing macromolecular crystals, but rather to replace the original CRS while helping to maintain crystal integrity and preserve parameters, particularly in terms of net osmotic pressure, dielectric constant, solubility of the corresponding biomacromolecules, and influence on the structural conformation of the biomacromolecules.

[0077] In another specific embodiment of the invention, the preferred salt group of the equivalent salt solution typically includes disodium 2,3-dihydroxysuccinate (sodium tartrate), (2R,3R)-2,3-dihydroxysuccinate (dipotassium tartrate), ammonium phosphate (ammonium phosphate), diammonium sulfate (ammonium sulfate), ammonium 2-hydroxypropyl-1,2,3-tricarboxylate (ammonium citrate), sodium acetate (sodium acetate), ammonium acetate (ammonium acetate), lithium sulfate, lithium chloride, sodium chloride, potassium chloride, sodium formate (sodium formate), sodium and potassium monophosphates, sodium and potassium diphosphates, etc. Phosphates and polyphosphates, sodium citrate (such as sodium 2-hydroxypropyl-1,2,3-tricarboxylate dihydrogen phosphate (mono-sodium citrate), disodium 2-hydroxypropyl-1,2,3-tricarboxylate hydrogen phosphate (disodium citrate), trisodium 2-hydroxypropyl-1,2,3-tricarboxylate (trisodium citrate), magnesium dicarboxylate, magnesium dichloride, magnesium sulfate, calcium chloride, disodium succinate (sodium succinate), cadmium(II) sulfate (cadmium sulfate), disodium malonate (sodium malonate), magnesium diacetate (magnesium acetate), zinc diacetate (zinc acetate), calcium diacetate (calcium acetate).

[0078] In one embodiment, the salt solution (crs) is a NaCl, MgCl2, or KCl solution, or contains NaCl, MgCl2, or KCl. Preferably, the concentration of NaCl, MgCl2, or KCl is between 0.1 M and 6 M. Preferably, the concentration of NaCl, MgCl2, or KCl is 1 M. Advantageously, the freshly prepared crs makes the process independent of the original crs. In a preferred embodiment, the same selected soaking solution is suitable for soaking crystals of different biomacromolecules, particularly different proteins.

[0079] Another embodiment of the present invention is a method for selecting the composition of an immersion solution, wherein the salt is sodium chloride, potassium chloride, magnesium diacetate (magnesium acetate), and lithium disulfate.

[0080] Preferably, when the freshly prepared salt solution of CRS is used as a soaking solution, the CRS is a mixture of PEGs, more preferably PEG400 and PEG3350.

[0081] In a preferred embodiment, the crs of the soaking solution is a NaCl, MgCl2 or KCl solution, and / or the cs is 3350, PEG 400, or a mixture of PEG400 and 3350.

[0082] In another embodiment, the crs does not contain any Cl salts, especially any NaCl.

[0083] One embodiment of the present invention is a method for selecting the composition of an immersion solution, wherein the crystallization solution CRS is obtained from the crystallization process of the crystal or is equivalent to it in terms of net osmotic pressure, dielectric constant, solubility of the corresponding biomacromolecule, and effect on the structural conformation of the corresponding biomacromolecule, and comprises pH and buffering substances, additives and precipitants or mixtures thereof, wherein the pH and buffering substances are selected from acetate buffer, TRIS buffer, HEPES buffer, dimethyl arsenate buffer, 1H-imidazole (imidazole) buffer and citrate buffer, wherein the precipitant is selected from volatile organic solvents, salts, polymers or non-volatile organic solvents, wherein the additives are selected from physiologically or biochemically relevant small molecules, chemical protectants, solubilizers and cleaning agents, compounds that reduce twinning, penetrants, cosolvents and structure-making agents, compounds that crosslink carboxyl and amino groups on protein surfaces, compounds that form electrostatic, covalent or hydrogen-bonded arrangements intended to stabilize biomacromolecule crystals, and substances or compounds that enhance nucleation and provide unique surfaces.

[0084] A preferred embodiment of the present invention is a method for selecting the composition of an immersion solution, wherein the crystallization solution CRS is obtained from the crystallization process of the crystal or is equivalent to it in terms of net osmotic pressure, dielectric constant, solubility of the corresponding biomolecule, and effect on the structural conformation of the corresponding biomolecule, and comprises pH and buffer substances, additives and precipitants or mixtures thereof, wherein the precipitant is selected from volatile organic solvents, salts, polymers or non-volatile organic solvents, wherein the volatile organic solvent is selected from ethanol, propan-1-ol, propan-2-ol, 2-propanol, 1,4-diol, etc. Alkane (di) Alkane), propan-2-one (acetone), 2-methylpropan-1-ol (isobutanol), butan-1-ol (n-butanol), 2-methylpropan-2-ol (tert-butanol), acetonitrile, methyl sulfoxide (dimethyl sulfoxide), oxepane-2-one (γ-butyrolactone), 4-methyloxepane-2-one (β-butyrolactone), wherein the equivalent salt solution contains salts selected from disodium 2,3-dihydroxysuccinate disodium salt (sodium tartrate), (2R,3R)-2,3-dihydroxysuccinate (dipotassium tartrate), ammonium phosphate (ammonium phosphate), diammonium sulfate (ammonium sulfate), ammonium 2-hydroxypropan-1,2,3-tricarboxylate (ammonium citrate), sodium acetate (sodium acetate), ammonium acetate (ammonium acetate), lithium sulfate, lithium chloride, sodium chloride, potassium chloride, sodium formate (sodium formate), sodium and potassium monophosphates, sodium and potassium The polymers are bisphosphates and polyphosphates, sodium citrate (such as sodium citrate dihydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (monosodium citrate), disodium hydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (disodium citrate), trisodium 2-hydroxypropyl-1,2,3-tricarboxylate (trisodium citrate), magnesium dicarboxylate, magnesium dichloride, magnesium sulfate, calcium chloride, disodium succinate (sodium succinate), cadmium(II) sulfate (cadmium sulfate), disodium malonate (sodium malonate), magnesium diacetate (magnesium acetate), zinc diacetate (zinc acetate), calcium diacetate (calcium acetate), wherein the polymers are selected from polyethylene glycol (preferably PEG400, PEG1000, PEG3350, PEG4000, PEG6000, PEG8000, PEG20000), Jeffamine T, Jeffamine M, 2-methoxyethanol (polyethylene glycol monomethyl ester), 2-hydroxyethyl octadecanoate (polyethylene glycol monostearate), and polyenamine, wherein the non-volatile organic solvent is selected from 2-methyl-2,4-pentanediol, hexane-2,5-diol (2,5-hexanediol), propane-1,3-diol (1,3-propanediol), polyethylene glycol (e.g., 400), and Jeffamine (e.g., 400).

[0085] Another preferred embodiment of the invention is a method for selecting the composition of an immersion solution, wherein the crystallization solution CRS is obtained from the crystallization process of the crystal or is equivalent to it in terms of net osmotic pressure, dielectric constant, solubility of the corresponding biomacromolecule, and effect on the structural conformation of the corresponding biomacromolecule, and comprises pH and buffering substances, additives and precipitants or mixtures thereof, wherein the additive group is selected from physiologically or biochemically relevant small molecules, chemical protectants, solubilizers and detergents, compounds that reduce twinning, penetrants, cosolvents and structure-forming agents, compounds that crosslink carboxyl and amino groups on protein surfaces, compounds that form electrostatic, covalent, or hydrogen-bonded arrangements aimed at stabilizing biomacromolecule crystals, and compounds for enhancing The category of substances or compounds that nucleate and provide a unique surface, wherein the small molecule of protein physiological or biochemical relevance is selected from 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiophene[3,4-d]imidazol-4-yl]valerate biotin, [[(2R,3S,4R,5R)-5-(6-aminopurine-9-yl)-3,4-dihydroxyoxacyclopentan-2-yl]methoxy-hydroxyphosphoryl]phosphonate hydrophosphate (adenosine triphosphate), [(2R,3S,4R,5R)-5-(6-aminopurine-9-yl)-3,4-dihydroxyoxacyclopentan-2-yl]methylphosphonate hydrophosphate (adenosine di ... R,5R)-5-(6-aminopurine-9-yl)-3,4-dihydroxyoxacyclopentan-2-yl]methyl dihydrogen phosphate (adenosine monophosphate), 3-[18-(2-carboxyethyl)-7,12-bis(vinyl)-3,8,13,17-tetramethyl-21H,23H-porphyrin-2-yl]ferric propionate (Fe-protoporphyrin IX or heme b), wherein the chemical protectant is selected from 2-mercaptoethanol (β-mercaptoethanol or BME) and (2S,3S)-1,4-bis(mercapto)but-2,3-diol (dithiothreitol or DTT), 3-[bis(2-carboxyethyl)phosphonyl]propionate (TCEP), and heavy metal ion scavengers such as 2-[2-[bis(carboxymethyl)amino] Ethyl-(carboxymethyl)amino]acetic acid (ethylenediaminetetraacetic acid or EDTA) and 2-[2-[2-[2-[2-[bis(carboxymethyl)amino]ethoxy]ethoxy]ethyl-(carboxymethyl)amino]acetic acid (EGTA), compounds that prevent microbial infection such as sodium azide (sodium azide), phenol, 1,1,1-trichloro-2-methylprop-2-ol (chlorobutanol), wherein the solubilizer and detergent are selected from quaternary ammonium salts, sulfobetaine, dissociating agents such as urea, and surfactants and detergent molecules such as (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-octaoxyoxacyclohexane-3,4,5-triol (n-octyl-β-d-glucoside), octyl-polyoxyethylene (octyl-POE), N,N-Dimethyldodecane-1-amine oxide (lauryl dimethylamine oxide), (2R,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6R)-6-dodecyloxy-4,5-dihydroxy-2-(hydroxymethyl)oxetane-3-yl]oxo-6-(hydroxymethyl)oxetane-3,4,5-triol (dodecyl-β-D-maltodextrin), sodium 1,4-bis(2-ethylhexyloxy)-1,4-dioxobut-2-sulfonate (bis(2-ethylhexyl)sulfosuccinate), dodecyl ammonium chloride (dodecyl ammonium chloride), polyethoxylated fatty acids, wherein the compound intended to reduce twinning is selected from ethanol, methyl sulfoxide (dimethyl sulfoxide or DMSO), acetone (acetone), 1,4-di-di-, Alkane (di) alkyl, but-1-ol (butanol), 2-methylpentane-2,4-diol (2-methyl-2,4-pentanediol or MPD), wherein the penetrant, co-solvent and structure-forming agent are selected from (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxetane-2-yl]oxo-6-(hydroxymethyl)oxetane-3,4,5- Triols (sucrose), (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxetane-2-yl]oxyoxetane-3,4,5-triols (trehalose) and other sugars, (2S)-pyrrolidine-2-carboxylic acid (proline), N,N-dimethylamine oxide (trimethylamine N-oxide or TM) AO), 2-aminoacetic acid (glycine), 2-(trimethylammonium)acetate (betaine), 2-aminoethanesulfonic acid (taurine), 2-(methylamino)acetic acid (sarcosine), wherein the category of compounds that crosslink carboxyl and amino groups on the protein surface is selected from molecules or aliphatic moieties containing diamino or dicarboxylic acids of different lengths carrying charged groups, wherein the category of compounds that form electrostatic, covalent, or hydrogen-bonded arrangements aimed at stabilizing the crystal through intermolecular crosslinking between proteins in the crystal is glutaraldehyde (glutaraldehyde), wherein the category of substances or compounds used to enhance nucleation and provide a unique surface is selected from PEG, Jeffamine, gels, such as gels used for cubic lipid phase crystallization and surfaces that promote epitaxial and heterogeneous nucleation, commercially available nucleating agents such as Crystallophore No. 1, Naomi nucleating agent, JBS Magical Triangle, Anderson-Evans polyoxotungstate (Na6[TeW6O24]·22 H2O) or buffer solution components that affect the solubility and aggregation properties of macromolecules, thus influencing their nucleation and growth properties, such as 2-aminoacetic acid (glycine), 3-[4-(3-sulfopropyl)piperazin-1-yl]prop-1-sulfonic acid (PIPPS), 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)prop-1,3-diol (Bis-Tris), 2-morpholino-4-ylethanesulfonic acid (MES), 2-[(2-amino-2- [Oxyethyl)-(carboxymethyl)amino]acetic acid (ADA), 2-[3-[[1,3-dihydroxy-2-(hydroxymethyl)prop-2-yl]amino]propylamino]-2-(hydroxymethyl)prop-1,3-diol (Bis-Tris propane), 3-morpholino-4-ylprop-1-sulfonic acid (MOPS), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-amino-2-(hydroxymethyl)prop-1,3-3-Diol (TRIS), 3-[4-(2-hydroxyethyl)piperazin-1-yl]prop-1-sulfonic acid (EPPS), 1H-imidazolium (imidazolium), 2-[bis(2-hydroxyethyl)amino]acetic acid (dihydroxyethylglycine), 2-(cyclohexylamino)ethanesulfonic acid (CHES), 3-(cyclohexylamino)prop-1-sulfonic acid (CAPS), (2R,3S,4S,5R)-2-(hydroxymethyl)-6-octaoxyoxacyclohexyl-3,4,5-triol (octyl) (2R,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6R)-6-dodecyloxy-4,5-dihydroxy-2-(hydroxymethyl)oxetane-3-yl]oxo-6-(hydroxymethyl)oxetane-3,4,5-triol (dodecyl maltodextrin), (2S,3S)-1,4-bis(mercapto)but-2,3-diol (DTT), and 3-[bis(2-carboxyethyl)phosphono]propionic acid (TCEP).

[0086] Another preferred embodiment of the present invention is a method for selecting the composition of an immersion solution, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is a polyhydroxy compound a or a mixture thereof, and the organic solvent (os) is a low-reactivity proton or aprotic liquid carbohydrate or a mixture thereof capable of dissolving small molecules. More preferably, the compatible solute (cs) is polyethylene oxide, and the organic solvent (os) is selected from (alkylated) sulfoxide, cyclic non-aromatic ether, straight-chain or branched monoaliphatic alcohol, alkylated formamide, or mixtures thereof. Most preferably, the compatible solute (CS) is selected from polyoxyethylene PEG200, PEG400, PEG600, or mixtures thereof, and the organic solvent (OS) is selected from dimethyl sulfoxide, 1,4-dioxane, etc. Alkane, dimethylformamide, methanol, ethanol, or mixtures thereof, The volume ratio of the compatible solute volume Vcs to the organic solvent volume Vos is the same, and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent Vos and the compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos;Vcs)).

[0087] Another preferred embodiment of the present invention is a method for selecting the composition of an immersion solution, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is a polyhydroxy compound or a mixture thereof, and the organic solvent is (alkylated) sulfoxide, cyclic non-aromatic ether, straight-chain or branched monoaliphatic alcohol, alkylated formamide or a mixture thereof. More preferably, the compatible solute (cs) is a polyol or a mixture thereof, and the organic solvent (os) is selected from (alkylated) sulfoxides, cyclic non-aromatic ethers, straight-chain or branched monoaliphatic alcohols, alkylated formamides or mixtures thereof. Most preferably, the compatible solute (cs) is selected from polyols propane-1,2,3-triol (glycerol), ethylene-1,2-diol (ethylene glycol), 2-methylpentane-2,4-diol (2-methylpentane-2,4-diol), (3R,4S,5S,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxacyclohexane-3,4,5-triol (1-glucosylglycerol), or mixtures thereof, and the organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dioxane, and other organic solvents. Alkane, dimethylformamide, methanol, ethanol, or mixtures thereof, The volume ratio of the compatible solute volume Vcs to the organic solvent volume Vos is the same, and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent Vos and the compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos;Vcs)).

[0088] Another preferred embodiment of the present invention is a method for selecting the composition of an immersion solution, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is a polyhydroxy compound a or a mixture thereof, and the organic solvent (os) is a low-reactivity proton or aprotic liquid carbohydrate or a mixture thereof capable of dissolving small molecules. More preferably, the compatible solute (cs) is a monosaccharide, disaccharide, trisaccharide, oligosaccharide, or polysaccharide, or a mixture thereof, and the organic solvent (os) is selected from (alkylated) sulfoxides, cyclic non-aromatic ethers, straight-chain or branched monoaliphatic alcohols, alkylated formamides, or mixtures thereof. Most preferably, the compatible solute cs is selected from sugars (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxacyclohexyl-2-yl]oxyoxacyclohexyl-3,4,5-triol (trehalose) or β-D-fructofuranosyl-α-D-glucopyranoside (sucrose) or mixtures thereof, and the organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dimethyl sulfoxide, etc. Alkane, dimethylformamide, methanol, ethanol, or mixtures thereof, The molar ratio of the organic solvent (os) to the compatible solute (cs) is the same (Mos / Mcs), wherein the molar ratio Mos / Mcs is from 1:10000 to 10000:1, preferably from 100:1 to 1:100, and more preferably from 10:1 to 1:10. The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent Vos and the compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 100:1 and 1:100, and most preferably between 85:15 and 1:1 (Vcrs:(Vos;Vcs)).

[0089] Another preferred embodiment of the present invention is a method for selecting the composition of an immersion solution, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is an L-amino acid or a D-amino acid or a mixture thereof, and the organic solvent (os) is a low-reactivity proton or aprotic liquid carbohydrate or a mixture thereof capable of dissolving small molecules. More preferably, the compatible solute (cs) is an L-amino acid or a mixture thereof, and the organic solvent is (alkylated) sulfoxide, cyclic non-aromatic ether, straight-chain or branched monoaliphatic alcohol, alkylated formamide or a mixture thereof. Most preferably, the compatible solute (cs) is selected from L-amino acid (2S)-pyrrolidine-2-carboxylic acid (proline), 2-aminoacetic acid (glycine), (6S)-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (ectoine), or (5S,6S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (hydroxyectoine), or mixtures thereof, and the organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dimethylaminosulfoxide, and other organic solvents. Alkane, dimethylformamide, methanol, ethanol, or mixtures thereof, The molar ratio of the organic solvent (os) to the compatible solute (cs) is the same (Mos / Mcs), wherein the molar ratio Mos / Mcs is from 1:10000 to 10000:1, preferably from 100:1 to 1:100, and more preferably from 10:1 to 1:10. The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent Vos and the compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 100:1 and 1:100, and most preferably between 85:15 and 1:1 (Vcrs:(Vos;Vcs)).

[0090] Another preferred embodiment of the present invention is a method for selecting the composition of an immersion solution, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is methylamine or a mixture thereof, and the organic solvent (os) is a low-reactivity proton or aprotic liquid carbohydrate or a mixture thereof capable of dissolving small molecules. More preferably, the compatible solute (cs) is selected from N,N-dimethylamine oxide (trimethylamine N-oxide or TMAO), 2-trimethylammonium acetate (trimethylglycine or betaine), 2-(methylamino)acetic acid (N-methylglycine or sarcosine), or mixtures thereof, and the organic solvent (os) is (alkylated) sulfoxide, cyclic non-aromatic ether, straight-chain or branched monoaliphatic alcohol, alkylated formamide, or mixtures thereof. Most preferably, the compatible solute (cs) is selected from N,N-dimethylamine oxide (trimethylamine N-oxide or TMAO), 2-trimethylammonium acetate (trimethylglycine or betaine), 2-(methylamino)acetic acid (N-methylglycine or sarcosine), or mixtures thereof, and the organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dimethylamine oxide, etc. Alkane, dimethylformamide, methanol, ethanol, or mixtures thereof, The molar ratio of the compatible solute to the organic solvent is the same, wherein the molar ratio is from 1:10000 to 10000:1, preferably between 100:1 and 1:100, and more preferably between 10:1 and 1:10. The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent Vos and the compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 100:1 and 1:100, and most preferably between 85:15 and 1:1 (Vcrs:(Vos;Vcs)).

[0091] Another preferred embodiment of the present invention is a method for selecting the composition of an immersion solution according to the present invention, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is PEG400, and the organic solvent (os) is dimethyl sulfoxide. The volume ratio of PEG400 (Vcs) to dimethyl sulfoxide (Vos) is the same, and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) PEG400 is 1:1000000 to 1000000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos:Vcs)).

[0092] Another preferred embodiment of the present invention is a method for selecting the composition of an immersion solution, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is ethylene-1,2-diol (ethylene glycol), and the organic solvent (os) is dimethyl sulfoxide. The volume ratio of ethylene-1,2-diol (ethylene glycol) Vcs to dimethyl sulfoxide (dimethyl sulfoxide) Vos is the same, and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) ethylene-1,2-diol (ethylene glycol) is 1:1000000 to 1000000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos:Vcs)).

[0093] Another preferred embodiment of the present invention is a method for selecting the composition of an immersion solution, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is propane-1,2,3-triol (glycerol), and the organic solvent (os) is dimethyl sulfoxide. The volume ratio of propan-1,2,3-triol (glycerol) Vcs to dimethyl sulfoxide (DMSO) Vos is the same, and the volume ratio is 1:1,000,000 to 1,000,000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) propion-1,2,3-triol (glycerol) is 1:1,000,000 to 1,000,000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos:Vcs)).

[0094] Another preferred embodiment of the present invention is a method for selecting the composition of an immersion solution, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is 2-methylpentane-2,4-diol (MPD), and the organic solvent (os) is dimethyl sulfoxide. The volume ratio of 2-methylpentane-2,4-diol (MPD) to dimethyl sulfoxide (DMS) is the same, and the volume ratio is 1:1,000,000 to 1,000,000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) 2-methylpentane-2,4-diol (MPD) is 1:1,000,000 to 1,000,000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos:Vcs)).

[0095] According to the present invention, a mixture of individual components of different soaking solution components selected from different groups and corresponding individual components of the same group of soaking solutions containing organic solvents, compatible solutes, crystallization solutions and water can be used in the array.

[0096] In one specific embodiment of the invention, the array according to the invention comprises individually soaked solutions physically arranged in each dimension dI of the array. The dimensions dI of the individually soaked solutions may or may not be physically arranged in a row of the array. It can also be arranged differently, as long as the position of each individually soaked solution can be determined to be bijective, i.e., in a one-to-one correspondence.

[0097] The subject of this invention is to use the array according to the invention to test the integrity of biomacromolecule crystals or to use the array according to the invention to test the tolerance of biomacromolecule crystals to: a) changes in the original growth environment caused by the introduction of organic solvents, compatible solutes (especially typical cryoprotectants), and dilution of the original crystallization buffer; b) stress caused by changes in pH and / or osmotic pressure of the crystal growth environment due to the introduction of, for example, organic solvents, compatible solutes, CRS diluents, and / or small molecules, or by other individual properties of the substances used (organic solvents, small molecules, etc. in the conditions evaluated in a); c) or using the array according to the invention for small molecule screening processes of biomacromolecule crystals; or d) using the array according to the invention to identify specific soaking solution compositions in the array suitable for small molecule screening processes of the biomacromolecule crystals; or e) using the array according to the invention to identify specific soaking solution compositions in the array suitable for reducing disorder in disordered regions of biomacromolecules.

[0098] In another aspect, the present invention relates to a method for selecting an immersion solution suitable for immersing biomacromolecules in crystalline form, wherein the solution comprises an organic solvent (OS), a compatible solute (CS), a crystallization solution (CRS), and / or water (W), the method comprising the following steps

[0099] 1) An immersion solution was prepared by mixing OS, CS, CRS, and W. 2) Transfer at least one crystal of the biomacromolecule to the solution prepared in step 1), or transfer the solution prepared in step 1) to a compartment containing at least one crystal of the biomacromolecule. 3) Control the crystal, 4) Select a suitable soaking solution for soaking the crystals.

[0100] In another aspect of the invention, the method is a method for selecting an immersion solution for immersing biomacromolecules in crystalline form, wherein the immersion solution comprises an organic solvent (OS), a compatible solute (CS), a crystallization solution (CRS), and / or water (W); the method includes the following steps

[0101] 1) Prepare a first (or baseline) series of 1n to xn individual soaking solutions by mixing os, cs, crs, and w, wherein: a) x is at least 2, b) Each soaking solution in the first series contains the same organic solvent (os), the same compatible solute (cs), and the same crystallization solution (crs). c) In every solution, Vos and Vcs are the same, or, in every solution, Mos / Mcs is the same. d) Vcrs and Vw vary in each solution. In solution 1n, Vcrs is the minimum Vcrs (Vcrsmin), and Vw is the maximum Vw (Vwmax). In solution xn, Vw is the minimum Vw (Vwmin), and Vcrs is the maximum Vcrs (Vcrsmax). When x>2, in any additional solution between 1n and xn, Vcrs varies between Vcrsmin and Vcrsmax, and conversely, Vw varies between Vwmin and Vwmax; 2) Transfer at least one crystal of the biomacromolecule from each compartment to x compartments, each compartment containing one of the solutions prepared in step 1); or transfer each solution prepared in step 1) to x compartments, each compartment containing at least one crystal of the biomacromolecule from its crystallization solution. 3) Control the crystals in each compartment; 4) Select a suitable soaking solution for soaking the crystals.

[0102] In a preferred embodiment, the first series of soaking solutions are distributed in each compartment to form a first dimension. Typically, the first dimension is a row or column of pores in a screening array.

[0103] In another embodiment, the method for selecting the composition of the soaking solution further includes the following steps:

[0104] 1) Prepare m series of 1n to yn individually soaked solutions by mixing os, cs, crs, and w, wherein m is at least 1, and wherein in each of the m series: a) y is at least 2, b) OS and CRS are the same as the first (or benchmark) series. c) Vos and Vcs are the same as in the first series, or Mos / Mcs are the same as in the first series. d) The cs is changed and is different from the cs of the first series. In each individual solution of each of the m series: e) Vos and Vcs remain unchanged, or Mos / Mcs remains unchanged. f) Vcrs and Vw vary in each solution. In solution 1n, Vcrs is the minimum Vcrs (Vcrsmin), and Vw is the maximum Vw (Vwmax). In solution yn, Vw is the minimum Vw (Vwmin), and Vcrs is the maximum Vcrs (Vcrsmax). When y>2, in any additional solution between 1n and yn, Vcrs varies between Vcrsmin and Vcrsmax, and conversely, Vw varies between Vwmin and Vwmax; 2) Transfer at least one crystal of the biomacromolecule from each compartment to y compartments, each compartment containing one of the solutions prepared in step 1); or transfer each solution prepared in step 1) to y compartments, each compartment containing at least one crystal of the biomacromolecule from its crystallization solution.

[0105] In a preferred embodiment, the method for selecting an immersion solution suitable for immersing biomacromolecules in crystalline form, wherein the immersion solution comprises an organic solvent (OS), a compatible solute (CS), a crystallization solution (CRS), and / or water (W); the method includes the following steps

[0106] 1) Prepare a first series of 1n to xn individual soaking solutions by mixing os, cs, crs, and w, wherein: a) x is at least 2, b) Each soaking solution in the first series contains the same organic solvent (os), the same compatible solute (cs), and the same crystallization solution (crs). c) In every solution, Vos and Vcs are the same, or, in every solution, Mos / Mcs is the same. d) Vcrs and Vw vary in each solution. In solution 1n, V crs For the minimum V crs (V) crs min), V w For the maximum V w (V) w max). In solution xn, V w For the minimum V w (V) w min), V crs For the maximum V crs (V) crs max). When x > 2, in any additional solution between 1n and xn, V crs In V crs min and V crs The values ​​of max and V vary, and conversely, V... w In V w min and V w Variations between max values; 2) Transfer at least one crystal of the biomacromolecule from each compartment to x compartments, each compartment containing one of the solutions prepared in step 1); or transfer each solution prepared in step 1) to x compartments, each compartment containing at least one crystal of the biomacromolecule from its crystallization solution. 3) Control the crystals in each compartment; 4) Select a suitable soaking solution for soaking the crystals; The compatible solute is selected from polyhydroxy compounds, amino acids, methylamine, and mixtures thereof. The organic solvent is selected from low-reactivity proton or aprotic liquid carbohydrates and mixtures thereof capable of dissolving small molecules, and the crystallizing solution crs is obtained from the crystallization process of the crystal or is equivalent to it in terms of net osmotic pressure, dielectric constant, solubility of the corresponding biomacromolecule, and effect on the structural conformation of the corresponding biomacromolecule, and contains pH and buffering substances, additives and precipitants.

[0107] In a preferred embodiment, Vcrsmin, Vcrsmax, Vwmin and / or Vwmax are the same and / or the same as the first (or reference) series in each of the m series.

[0108] In another embodiment, the number of individual soaking solutions y for each of the m series is the same as the number of individual soaking solutions x for the first series; and / or in any additional solution between 1n and xn and / or in any additional solution between 1n and yn, Vcrs gradually varies between Vcrsmin and Vcrsmax, and conversely, Vw gradually varies between Vwmin and Vwmax.

[0109] In a preferred embodiment, in any additional solution between 1n and yn, the increment of Vcrs between Vcrsmin and Vcrsmax and the increment of Vw between Vwmin and Vwmax are the same in the first series and in each of the m series.

[0110] In a preferred embodiment, the m series of solutions are distributed in each compartment to form a second dimension. Typically, if the first dimension is represented by a row of wells in a standard sieve or crystallization array (e.g., containing the first series of solutions), then the other rows of wells (e.g., containing the m series of solutions) will create columns of wells, which will become the second dimension. Conversely, if the first dimension is represented by a column of wells in a standard sieve or crystallization array (e.g., containing the first series of solutions), then the other columns of wells (e.g., containing the m series of solutions) will create rows of wells, which will become the second dimension.

[0111] In another embodiment, the crs is a salt solution that is freshly prepared, i.e., not derived from the crystallization process or the solution used to grow the crystals. In this embodiment, the freshly prepared crs typically consists of a salt solution that is not the original crs but is equivalent and maintains a certain level of ionic strength or buffering capacity while preserving crystal integrity. In a preferred embodiment, the crs is a salt solution different from the original crystallization solution used for the nucleation, growth, and / or storage of macromolecular crystals, wherein the salt of the crs is any salt suitable for the crystallization of biomacromolecules.

[0112] Typically, the newly prepared CRS is referred to as an equivalent salt solution, preferably a Cl solution, particularly NaCl, MgCl2, or KCl. In a preferred embodiment, when the salt solution of the newly prepared CRS is used as a soaking solution, the CRS is a mixture of PEGs, more preferably PEG400 and PEG3350. In a different embodiment, the CRS is a salt solution different from the original crystallization solution used for the nucleation, growth, and / or storage of macromolecular crystals, wherein the salt of the CRS is not a chloride (Cl) salt, and particularly not NaCl.

[0113] In another embodiment, the cs is selected from PEG400, MPD, EG and / or glycerol. The cs is preferably a mixture of PEGs, more preferably PEG400 and PEG3350, or a mixture of MPD and PEG, especially a mixture of MPD and PEG3350.

[0114] In a preferred embodiment, the OS is DMSO.

[0115] Preferred V crs min is 0%.

[0116] Preferred V w min is 0%.

[0117] The preferred volume V of OS os Greater than 0.5%, preferably at least 10%, or at least 15%, or at least 20%, more preferably between 5% and 25%.

[0118] The preferred cs volume is at least 5%, more preferably 10%, or at least 20%, at least 30%, at least 40%, at least 50%, more preferably between 5% and 80%, and most preferably 10% to 60%.

[0119] In a preferred embodiment, the soaking solution comprises crs, w, os, and / or cs. In one embodiment, the percentage of the total volume (VT) of each solution in the first series is VT = Vcrs + Vw + Vos + Vcs = 100%. In this preferred embodiment, os may or may not contain small molecules.

[0120] In terms of Nos, Ncs, Ncrs, and Nw, "N" always refers to the number of moles of the relevant component. NT refers to the total number of moles of the component in the solution, i.e., NT = Ncrs + Nw + Nos + Ncs.

[0121] In a preferred embodiment, the ratio of the number of moles of the compatible solute to the total number of moles of the components (Ncs / NT) is at least 0.05, more preferably at least 0.1, or at least 0.2, or at least 0.3, or at least 0.4, or at least 0.5, more preferably between 0.05 and 0.8, and most preferably between 0.1 and 0.6.

[0122] Typically, in the preferred embodiment, an immersion solution is considered suitable for immersing the crystals when the quality of the crystals is sufficient to allow for other uses and / or analysis of the crystals. Examples of such uses and / or analyses include hit verification in electron microscopy, neutron diffraction, small molecule screening, other screening methods such as high-throughput screening (HTS) or biophysical screening, biotechnological applications such as chromatography, biosensors, or crystal applications in materials science (hybrid materials). In one preferred embodiment, the immersion solution is selected if it is suitable for a small molecule screening process.

[0123] In one specific embodiment of the invention, the specific immersion solution is preferably controlled by means of X-ray diffraction and / or visual control, according to defined criteria commonly used by experts in the relevant crystallography field (particularly, in X-ray diffraction: maximum resolution should be less than 2.8 Å, preferably less than 2.5 Å; low mosaicism, typically less than 0.8°; no or minimal ice rings; high-quality electron density as judged by an experienced crystallographer; for visual control: no or minimal fractures or cracks; no or minimal dissolution; sharp crystal edges; color in polarized light, etc.). A suitable immersion solution is then selected to immerse the crystal, thereby generating a signal in subsequent analysis. In another additional embodiment of the invention, the steps of controlling the crystal and selecting a suitable solution are performed based on defined quality criteria (such as resolution, mosaicism, electron density, and the presence of diffraction artifacts from unwanted ice crystals, salt crystals, or other crystals grown from components such as the immersion solution composition and crystal twin components).

[0124] In this invention, the crystal control step can be performed by any method available to a person skilled in the art to assess the presence and / or aspects of the crystal and its suitability for further analysis (such as X-ray diffraction analysis). In a preferred embodiment, crystals from different biomacromolecules (preferably proteins) are tested in the same solution. Specifically, the crystal control step refers to assessing the tolerance of one or more types (i.e., from different biomacromolecules) of biomacromolecule crystals in a specific immersion solution in terms of their presence, and / or measuring the effect of the immersion solution on the crystal integrity / stability. Preferred methods are X-ray diffraction, or neutron diffraction at an internal source or synchrotron, and visual analysis of the crystals by means of UV / VIS and IR spectroscopy.

[0125] In one preferred embodiment, the decision to select the immersion solution is made after visual control and / or X-ray evaluation. In one preferred embodiment, all types of crystals or crystal residues are controlled by X-ray analysis, because sometimes poorly shaped crystals can diffract well. In one preferred embodiment, only voids where the crystals have completely dissolved or split are discarded.

[0126] In a preferred embodiment, more than one crystal is controlled. In a most preferred embodiment, crystals derived from at least two different biomacromolecules are controlled in the same solution composition.

[0127] In a preferred embodiment, the biomacromolecule is a protein, preferably a binding protein, and more preferably a receptor protein.

[0128] In another preferred embodiment, the macromolecule is an RNA molecule. In a most preferred embodiment, the RNA molecule is messenger RNA. More preferably, the mRNA contains a riboswitch, and even more preferably, the mRNA contains an aptamer.

[0129] The visual inspection preferably examines how the appearance of the crystal changes over time.

[0130] In a preferred embodiment, the composition of the soaking solution is selected after visual analysis of the crystal. Examples of unsuitable soaking solution compositions are those that cause complete dissolution or severe damage to the crystal, such as breakage, and are therefore discarded. X-ray analysis can then be performed on the first set of soaking solutions selected after visual control. All control and selection parameters can be set using only common general knowledge. The method according to the invention for controlling the crystal and selecting a suitable soaking solution does not require any specific knowledge beyond the common general knowledge of experts in protein crystallization. Parameters of the control method, such as maximum resolution, low mosaicism, intact crystal structure, electron density quality, absence of twins due to unwanted ice crystals, and absence of diffraction signals, are set to allow for standard control of crystal integrity, after which a suitable soaking solution is selected.

[0131] In a preferred embodiment, data collected during the crystal control step is analyzed to select an immersion solution. The immersion solution is selected to improve the crystal's stability over time, or at least has no adverse effect on the crystal or only minimal adverse effects. The selection criteria can be determined by a crystallographer using common knowledge.

[0132] In a preferred embodiment, the crs is a salt solution different from the original crystallization solution used for the nucleation, growth, and / or storage of macromolecular crystals. Using freshly prepared crs makes the method independent of the original crs. In a preferred embodiment, the salt of the crs is any salt suitable for crystallizing biomolecules. Preferably, the salt of the solution is NaCl, MgCl2, or KCl. In a different embodiment, the salt is not a chloride (Cl) salt, and particularly not NaCl. In a preferred embodiment, the same soaking solution is suitable for soaking crystals of different biomolecules (especially different proteins).

[0133] In a preferred embodiment, more than one crystal is controlled. In a most preferred embodiment, crystals derived from at least two different biomacromolecules are controlled in the same solution composition.

[0134] In a preferred embodiment, cr, os, cs and / or w contain small molecules, or preferably, os contains small molecules.

[0135] In a preferred embodiment, the OS contains small molecules as defined below, particularly small molecule fragments or molecular probes.

[0136] In one preferred embodiment, the concentration of the small molecule used for soaking is 10 times the binding constant of the corresponding small molecule to the corresponding biomacromolecule. In one preferred embodiment, a small molecule or mixture of small molecules is used, typically in the micromolar to molar range, preferably in the range of 5 mM to 500 mM per individual soaking solution. The concentration of the drug-sized small molecule is at least about 5-20 mM. The concentration of the fragment is preferably at least 10-50 mM. In one preferred embodiment, particularly in OS, a high concentration of small molecules exceeding 500 mM and at most 1 M is used (provided it does not adversely affect the crystals).

[0137] When the soaking solution contains small molecules, a preferred embodiment involves further stress testing of the crystals to assess their tolerance to changes introduced by the addition of small molecules. Such stress conditions occur upon the introduction of small molecules that alter the pH, or are caused by osmotic pressure changes induced by high concentrations of small molecules. Therefore, under previously identified soaking conditions containing biomacromolecules, the tolerance of the biomacromolecules to pH changes is tested by adding different concentrations of hydrochloric acid fragments and fragments and / or molecular probes.

[0138] In another aspect of the invention, the method is a method for small molecule screening of biomacromolecule crystals, wherein the screening is carried out in an soaking solution selected by the method for selecting soaking solutions according to the invention.

[0139] In another aspect, the present invention is an array arranged to perform the methods described above.

[0140] The present invention also relates to an soaking solution that can be obtained by any of the methods described above for selecting the composition of the soaking solution. In a preferred embodiment, the soaking solution of the present invention has a Voss greater than 5%, preferably greater than 10%.

[0141] In a more preferred embodiment, the soaking solution obtained by any of the methods described above for selecting the composition of the soaking solution is free of Cl, especially NaCl.

[0142] The present invention also relates to the use of the selected soaking solution for small molecule screening of biomacromolecule crystals.

[0143] In a preferred embodiment, several solutions are preferably tested simultaneously, and more than one solution is selected, thereby defining the volume / percentage range of each component of the tested solution.

[0144] In another specific embodiment of the present invention, the biomacromolecule crystals are incubated in an immersion solution containing small molecules or a mixture of small molecules for less than 1 minute to 72 hours, preferably 24 hours.

[0145] In another specific embodiment of the present invention, the biomacromolecule crystal is vitrified at low temperature or subjected to direct X-ray measurement at room temperature.

[0146] In an additional embodiment of the invention, the biomacromolecule crystals are preferably stored at low temperatures in liquid nitrogen and / or helium, or the biomacromolecule crystals are directly collected by imaging techniques including X-ray diffraction, electron diffraction, and neutron diffraction.

[0147] In this invention, a single soaking solution composition suitable for a small molecule screening process of the biomacromolecule crystals means that if the soaking solution allows for a “hit-and-pick” process, it is deemed “suitable”, particularly for small molecule screening processes, wherein the binding of a specified small molecule to a biomacromolecule crystal is defined as a “hit”.

[0148] In a preferred embodiment, if the composition of the soaking solution is capable of soaking fragments and small molecule ligands into the biomacromolecule crystal, resulting in a maximum possible resolution of less than 2.8 Å, preferably less than 2.5 Å, in the case of molecular probes and fragments, and less than 3.5 Å in the case of drug-sized molecules, then it is suitable for small molecule screening processes using the biomacromolecule crystal. Ideally, the biomacromolecule crystal exhibits minimal mosaicism, typically less than 0.8°; and displays an electron density, which is then evaluated, for example, by an experienced crystallographer.

[0149] The subject of this invention is a method according to the above invention, wherein the method is automated.

[0150] In another embodiment, the subject matter of the invention relates to any of the selected soaking solutions in the above embodiments of the method for selecting the composition of the soaking solution according to the invention. In a preferred embodiment, the soaking solution contains at least 5% os.

[0151] In another embodiment, the subject of the invention is the use of any of the selected soaking solutions in the above embodiments of the method for selecting the composition of the soaking solution for small molecule screening of biomacromolecule crystals.

[0152] In another embodiment, the subject of the invention is an array arranged to perform the method of the invention and an array containing a solution prepared as described in preparation step 1 of the method of the invention, particularly the method described above, or an array containing an soaking solution selected by the method of the invention for selecting the composition of the soaking solution.

[0153] Another embodiment of the invention is an array arranged to perform the method, wherein the array comprises a first dimension of at least two separate soaking solutions (1n to xn) and a second dimension of at least two separate soaking solutions (1m to ym), wherein each of the soaking solutions is located in a separate compartment of the array, wherein each of the soaking solutions comprises an organic solvent (os), a compatible solute (cs), a crystallization solution (crs), and water, wherein in a series of soaking solutions along the first dimension, the ratio of the volume of the compatible solute Vcs to the volume of the organic solvent Vos is the same, or, in a series of soaking solutions along the first dimension, the ratio of the volume of the organic solvent (os) to the volume of the compatible solute (cs) is... The molar ratio Mos / Mcs is the same, wherein in a series of soaking solutions in the first dimension, the individual soaking solutions in the first dimension each contain the same organic solvent and the same compatible solute, wherein in the series of individual soaking solutions in the first dimension, the ratio of water volume Vw to crystallization solution volume Vcrs is varied, wherein one of the series of individual soaking solutions in the first dimension has a minimum or zero Vcrs and a maximum Vw, and another individual soaking solution in the first dimension has a minimum or zero Vw and a maximum Vcrs, wherein the other individual soaking solutions in the series in the first dimension take Vcrs and Vw values ​​between the two aforementioned values.

[0154] Another embodiment of the invention is an array in which 1n to xn defines the number of individual soaking solutions in the range of 1 (1n) to 1×106 (xn), wherein the numbers include, but are not limited to, 1×106 or 1×105, 1×104 or 768 or 384 or 96 or 48 or 24, however, the arrangement of the array determines the number of individual soaking solutions.

[0155] A preferred embodiment of the present invention is an array comprising a second dimension (dII) of a series of individual soaking solutions of biomacromolecule crystals, wherein the compatibility solute varies in the second dimension and depends on the compatibility solute used in the series of soaking solutions in the second dimension. The ratio of the volume of the compatibility solute (Vcs) to the volume of the organic solvent (Vos) or the molar ratio of the organic solvent (os) to the compatibility solute (cs) (Mos / Mcs) may be different.

[0156] Another preferred embodiment of the invention is an array in which, in each of the series of said individual soaking solutions, the ratio of the volume of the compatible solute Vcs to the volume of the organic solvent Vos is the same, said volume ratio being from 1:1,000,000 to 1,000,000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos).

[0157] Another preferred embodiment of the invention is an array in which, in each of the series of individual soaking solutions, the molar ratio of organic solvent (os) to compatible solute (cs) is the same (Mos / Mcs), said molar ratio (Mos / Mcs) being 1:10000 to 10000:1, preferably between 100:1 and 1:100, more preferably between 10:1 and 1:10.

[0158] Another embodiment of the present invention is the use of an array for obtaining the solution described in the above embodiments.

[0159] definition

[0160] Definition of a compartment

[0161] A compartment is a container that refers to a separate part or component in an array specifically designed for culturing or screening. In this invention, a compartment can be a single pore of an array, or it can also refer to any container or part of an array or component in which experiments can be performed. This includes commercially available and custom-made (i.e., printed in a 3D printer, etc.) plates of any suitable material and any type of pore for any other device that can be used for immersion and / or screening. The term "compartment" can also refer to a portion or organelle of a living cell or preserved cell (including, but not limited to, mammalian, insect, and bacterial cells), or additionally to a (bio)chip, foil, tubular, or tube of various shapes made of any suitable material.

[0162] Array definition

[0163] An “array” is one or a series of compartments or containers, or includes an arrangement of elements (i.e., crystals) in a pattern on a solid carrier and / or container. In this invention, an array can also refer to a single container / compartment. When an array is a single compartment, it can also refer to a tube or a hole. While the pattern of an array is typically arranged in two-dimensional space, in particular, standard screening arrays or commercially available arrays for high-throughput screening consist of numerous individual compartments or holes forming rows on two different vertical axes. The pattern can also be arranged in one-dimensional space (e.g., a tube or a single hole or a single row of holes) or three-dimensional space (e.g., at least two arrays with two dimensions superimposed). However, this invention is not limited to simple spatial organization. For example, an information technology system can be used to generate logical arrays in which the distribution, size, and location of corresponding components can be tracked (i.e., in a table / form), but cannot be characterized by simple spatial organization. Furthermore, the term array can also refer to different forms, including “chip” or “biochip.” Here, the array can be arranged in or on an array at a low density (i.e., defined as two to ten distinct addressable sites) to a high density (i.e., more than one thousand addressable sites). It should be noted that the shape and size of the array chip or biochip can be irregular and not limited by any geometry, but a typical chip array is rectangular. The configuration of the array can include bundling, mixing, and / or homogenizing multiple addressable sites suitable for, for example, high-throughput processing and robotic reagent delivery. The spatial organization of the array also allows for the detection and / or quantification of specified signals, including but not limited to chemiluminescence, X-rays, confocal or deflection focusing, and laser illumination. Array forms include any suitable form for culturing and soaking biomolecular crystals, including but not limited to microarrays, microchips, biomolecular arrays attached to multilayer plates, and living organisms such as insect cells, bacterial cells, and mammalian cells (“in vivo crystallization”). Other examples of array forms include serial crystallography, foil, and tubular devices on or within which crystals can be X-ray measured.

[0164] Definition of a series of individual soaking solutions

[0165] A series of individual soaking solutions refers to at least two individual soaking solutions that share several common characteristics. Specifically, the series of individual soaking solutions preferably has at least one common component, such as the same organic solvent (OS), compatible solute (CS), and / or crystallizing solution (CRS). Common characteristics may include the same volume of one, two, or three or more components, the volume ratio of some components to one or more components, and / or the number of individual solutions.

[0166] When more than one series is prepared, one is called the "first series," but it can also be called the "reference" series. This series is conveniently referred to as "first" because it is typically distributed in the first row of the array. However, the so-called first series can also be called the "reference" or "reference" series, and is not necessarily distributed in the first row or first column of holes of the array. In practice, this reference series serves as a reference for preparing additional series according to the method of the invention. When more than one series is prepared, the so-called first series or reference series can therefore be any of the series that will subsequently be used as a reference for the rule-based method of the invention.

[0167] In a preferred embodiment, at least two separate soaking solution series refers to the composition of at least two soaking solutions comprising volumes of an organic solvent, a compatible solute, distilled water, and a crystallization solution, wherein a different compatible solute is used in each series.

[0168] In one embodiment, in each series, the crystallization solution, or a crystallization solution of several times its original concentration, is diluted in a continuous dilution manner, while the volume of distilled water is increased proportionally. In each series, the volumes of the compatible solute and the organic solvent, as well as the ratio between all volumes, remain constant.

[0169] Definitions of proteins, polypeptides, and RNA

[0170] Polypeptide: The term "polypeptide" refers to any oligomeric arrangement of amino acids, which are usually, but not only, covalently linked by peptide bonds and appear in different lengths. Generally, the term "polypeptide" refers to an amino acid sequence having fewer than 70 amino acids and a molecular weight of less than 70 kDa. Polypeptide sources include natural or non-natural sources, or combinations thereof, including naturally occurring polypeptides, polypeptides produced through recombinant molecular genetics, polypeptides derived from cells or translation systems, or polypeptides produced by cell-free synthetic methods. The amino acid sequence of a polypeptide determines its structure and is not limited to a full-length sequence, but can also be a partial or complete sequence. Generally, the term "peptide" refers to a small polypeptide consisting of 2-25 amino acids in length, while a natural polypeptide is a polypeptide whose amino acid residue sequence is identical to that found in nature (e.g., wild-type polypeptides). Furthermore, natural polypeptides can have a natural source, can be isolated from their natural source (i.e., plant cells or tissues), or can be genetically engineered using recombinant genetics. Depending on the naturally occurring wild-type polypeptide, post-translational modifications may or may not be present in a natural polypeptide. The term polypeptide can also refer to a polypeptide fragment. As used herein, this term refers to any contiguous subset of the amino acid sequence of a full-length polypeptide. A polypeptide fragment or portion can be isolated from any domain of the polypeptide and is not limited to a specific length from about 4 amino acids up to the full-length polypeptide sequence. Furthermore, a polypeptide may or may not have any specified biological or synthetic activity. The term "protein" refers to a polymer of amino acids linked together by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. As stated in the definition of polypeptide, proteins can have natural, recombinant (i.e., biotechnically produced in genetically modified organisms or transiently transfected cell cultures with or without mutations), synthetic, or any combination thereof. Furthermore, the term "protein" refers to a single molecule or a complex of multiple molecules (a complex of a protein with, for example, other peptides, polypeptides, and proteins). The term protein may also apply to amino acid polymers containing one or more amino acid residues of an artificial chemical analog of the corresponding naturally occurring amino acid. Additionally, the term protein can also refer to prions, which are misfolded proteins capable of transferring their aberrant shape to harmless variants of the same protein.

[0171] RNA is a polymeric biomolecule composed of ribonucleotides (nitrogenous bases attached to ribose sugars and linked by phosphodiester bonds), forming chains of varying lengths and conformations (e.g., convex and helical, circular, etc.). Nitrogenous bases include adenine, guanine, cytosine, and uracil. RNA can be broadly classified based on the length of its nucleotide chain, including small RNAs (less than 200 nucleotides) and long or large RNAs (more than 200 nucleotides). The small RNA group includes, but is not limited to, transfer RNA (tRNA), 5S ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA), while the long or large RNA group includes, but is not limited to, long non-coding RNA (incRNA) and messenger RNA (mRNA), especially mRNAs containing ribosomes (see definition in the next paragraph). However, the term RNA can also refer to the genetic material (2 to 20 kb) of RNA viruses, including double-stranded RNA, single-stranded RNA (+ / -), isometric particles with single-stranded RNA, and pseudoviridae (i.e., Ebola virus, SARS, influenza virus, etc.). Furthermore, the term RNA can refer to viroids or virusoids / satellite viruses. Viroids are single-stranded, covalently closed circular RNA molecules characterized by their small size (250-400 nucleotides). They do not encode any proteins and lack a capsid (but contain a protein coating). Viroids or satellite viruses also refer to circular subviral particles with single-stranded RNA that rely on viruses for replication and encapsulation. The genome of a virusoid typically consists of several hundred nucleotides (200-400).

[0172] Riboswitches are regulatory segments in messenger RNA molecules that bind small molecules, thereby causing changes in the production of proteins encoded by the mRNA. Conceptually, they are generally divided into two parts: an aptamer and an expression platform. The aptamer directly binds to the small molecule, and the expression platform undergoes structural changes in response to changes in the aptamer. The expression platform regulates gene expression. A preferred embodiment of the present invention is wherein the biomacromolecule originates from mRNA containing an aptamer that includes a riboswitcher.

[0173] Definition of binding site: A macromolecule binding site is any location on a macromolecule (such as a protein or mRNA) that specifically binds to another molecule. This binding complex is called a ligand and can include other proteins that cause protein-protein interactions or small molecules containing, for example, enzyme substrates, hormones, allosteric regulators, second messengers, and inhibitors. For example, a binding event can lead to a conformational change in the corresponding macromolecule, thereby altering its function or activity (e.g., in proteins) and riboswitch in mRNA. Most of these binding events are non-covalently reversible (temporary), with fewer being covalently reversible or irreversible. Types of binding sites include active sites (orthomeric sites) and allosteric sites.

[0174] Active sites bind to substrates that induce chemical reactions and inhibitors that suppress those reactions. Allosteric sites are regulatory sites that bind ligands that enhance or inhibit the function of macromolecules.

[0175] Definition of water: Water refers to an inorganic substance that typically contains one oxygen atom and two hydrogen atoms linked by covalent bonds. In this invention, the term water refers to, but is not limited to, four different types of water used, for example, in laboratories. Type I water is ultrapure water with low levels of bacteria and organic matter, a maximum sodium content of 1 μg / L, a maximum chlorine content of 1 μg / L, a maximum total silica content of 3 μg / L, and a resistivity typically >18 MΩ. cm. Type II water is pure water with a resistivity >1 MΩ. The water has low levels of bacteria and organic matter, with a maximum sodium content of 5 μg / L, a maximum chlorine content of 5 μg / L, and a maximum total silica content of 3 μg / L. Here, deionization or (electro)ion exchange removes ions from the reverse osmosis water using synthetic resins. Type III water has a lower purity than Type I and Type II water and is produced directly from tap water using a reverse osmosis system and carbon filtration. Type III water has a resistivity >4 MΩ. The highest sodium content was 10 μg / L, the highest chlorine content was 10 μg / L, and the highest total silica content was 500 μg / L. Finally, the resistivity of type IV water was >200 MΩ. cm, with a maximum sodium content of 50 μg / L, a maximum chlorine content of 50 μg / L, and no limit on total silica content. However, the term "water" can also refer to water supply or so-called raw water or drinking water, the quality of which depends on its source. As used herein, the term "water" refers to examples such as tap water, desalinated water, sterile filtered water, distilled water, DNase / RNase-free water (e.g., DEPC water), deionized water, reverse osmosis water, industrial water, and heavy water. It should be noted that the term "water" refers to any kind of water suitable for the purposes of this invention.

[0176] Definition of immersion solution

[0177] A soaking solution is any solution that serves as the liquid environment for biomacromolecule crystals, but whose composition differs from the original environment in which the biomacromolecule crystals nucleate and grow. Typically, it originates from the original crystal growth environment and contains additional organic solvents or compatible solutes and other components. The components of the soaking solution derived from the original growth environment may have the same concentration as their individual components, a multiple of that concentration, or be diluted.

[0178] Immersion solutions are commonly used to introduce small molecules, such as molecular probes, fragments, and drug-sized molecules, into crystals to elucidate the binding events of these small molecules in subsequent diffraction experiments. Furthermore, immersion solutions are used to introduce heavy atoms into biomacromolecule crystals, thereby obtaining phases with previously unknown structures during subsequent diffraction experiments. They are also used to introduce cryoprotectants to modulate low-temperature stress tolerance for the preparation of crystals for biotechnological applications (e.g., chromatography or biosensors) and / or to improve crystal quality. The volume of immersion solutions typically ranges from μm to ml, usually between 1 and 10 μl. In automated applications, nanoliter-scale volumes can be adjusted accordingly.

[0179] The volume of the immersion solution alone is in the range of picoliters to milliliters.

[0180] Unless the volume is explicitly expressed in a standard volume unit, such as µm or ml, "V" (as in Vos, Vcs, Vcrs, and Vw) always refers to the proportion of the relevant component in the total volume (VT) of the solution, i.e., 100%. For example, Vos refers to the percentage / proportion of organic solvent (os) in the total volume of the solution. For instance, Vos=10 means that os accounts for 10% of the total volume of the solution, while Vos=10 µl means 10 µl of os solution.

[0181] In a preferred embodiment, VT is VT = Vcrs + Vw + Vos + Vcs, meaning the solution consists of a mixture of crs, w, OS, and cs. In some embodiments, Vcrs and / or Vw may be 0% or not. Therefore, some solutions may contain only OS and cs, or OS, cs, and crs, or OS, cs, and water.

[0182] Preferred V crs min is 0%.

[0183] Preferred V w min is 0%.

[0184] Preferred OS volume V os The content is greater than 5%, preferably at least 10%, at least 15%, or at least 20%, more preferably between 5% and 25%. In another embodiment, the Vos is at least 0.5%.

[0185] The preferred cs volume is at least 5%, more preferably 10%, or at least 20%, at least 30%, at least 40%, at least 50%, more preferably between 5% and 80%, and most preferably 10% to 60%.

[0186] Small molecules, especially molecular probes, fragments, and drug-sized small molecules, are defined.

[0187] In molecular biology and pharmacology, the term "small molecule" refers to any low-molecular-weight organic compound, typically ranging from 100 to 500 Da, but in special cases it can extend to approximately 2000 Da (e.g., in the case of macrolides or proteolytic targeted chimeras (PROTACs), in contrast to high-molecular-weight compounds such as polymers like RNA, DNA, proteins, or polysaccharides. Small molecules even include monomeric or oligomeric components of RNA, DNA, proteins (peptides), and polysaccharides, such as ribonucleotides and deoxyribonucleotides, amino acids, monosaccharides, or oligosaccharides. Many small molecules regulate biological processes or are otherwise related to biological systems. Therefore, they can function as agonists and antagonists, as inhibitors or mediators of protein-protein interfaces, proximity, or interactions, and as regulators of biophysical properties such as osmotic pressure. They can have a variety of functions, such as (but not limited to) signal transduction, metabolism, osmotic pressure regulation, and cryoprotection. They can be applied to applications such as drugs, molecular probes, starting points for drug discovery, leading compounds, clinical candidates, pesticides, cosmetics, food chemicals, fragrances, fungicides, and many others. Their sources can be natural, such as secondary metabolites, or synthetic, such as drugs, and they can have beneficial effects (e.g., therapeutics) or adverse effects (e.g., poisons). In drug discovery, small molecules are used to screen for interactions with target macromolecules; they may or may not have binding affinity for the corresponding target, and they may or may not alter biological processes. They can be used as molecular probes or small molecule fragments.

[0188] A molecular probe is a small molecule (30-120 Da) linked with functional groups (e.g., amino, acid, hydroxyl, etc.) and used in molecular sciences such as biochemistry, structural biology, chemistry, or medicinal chemistry to study the properties of other molecules, especially biologically relevant macromolecules (RNA, DNA, proteins, peptides, polysaccharides), and to explore their interaction characteristics. Therefore, a molecular probe can be a group of atoms or molecules used in molecular biology or chemistry to study the properties of other molecules or structures. If a measurable property of the molecular probe changes when it interacts with an analyte (e.g., a change in absorbance), the interaction between the probe and the analyte can be studied. Radioactive DNA or RNA sequences are used in molecular genetics to detect the presence of complementary sequences through molecular hybridization. In computational methods, a molecular probe can even be an artificial construct that primarily reflects a property such as a hydrogen bond donor or a lipophilic probe. If a measurable property of the applied molecular probe changes when it interacts with an analyte (e.g., a change in absorbance or interaction geometry), the interaction between the probe and the analyte can be studied. In molecular sciences such as structural biology (e.g., crystallography), molecular probes are used to map the properties of binding sites on macromolecular targets due to the presence of: polarization effects induced by hydrophobic and hydrophilic H-bonding modes, leading to changes in protonation states, conformational changes, and induced molecular adaptations. Furthermore, molecular probes provide information about putative pharmacophore patterns, which helps determine which group of small molecules should be used for screening. Additionally, molecular probes can be used for stress testing, such as assessing the tolerance of macromolecules or macromolecular crystals to, for example, organic solvents or pH changes. Molecular probes can indirectly study the properties and structures of compounds that might be difficult to study directly. The choice of molecular probe will depend on the compound or structure being studied and the properties of interest.

[0189] The term "small molecule fragment" refers to a small molecule (e.g., size ≤250 kDa) that typically has an affinity ranging from a low single-digit micromolar to a double-digit millimolar range. Small molecule fragments, typically 120–250 Da in size, serve as a starting point for optimization through chemical synthesis, for example, for the production of ligands such as inhibitors, agonists, or antagonists. These small molecule fragments can be, for example, peptides, any organic molecule or natural product, short nucleic acid sequences (e.g., DNA or RNA types), and / or any other ligands, including solvent molecules and cryobuffer components capable of binding to macromolecular targets. A "target" can be any macromolecular system with a biological function or a macromolecular crystal system based on amino acids, nucleic acids, and additional sugar or lipid moieties as monomer building blocks; for example, enzymes, soluble proteins, or membrane-bound proteins such as soluble or membrane-bound receptors or ion channels, RNA and DNA molecules, or glycosylated macromolecules.

[0190] "Drug-size molecules" are organic compounds ranging from 250 to 2000 Da that may or may not exert therapeutic effects as drugs or lead candidates. Those that do exert such therapeutic effects serve to regulate some biological activity, such as the activity of macromolecules, for example, as agonists, antagonists, or catalysts, such as in targeted protein degradation. Drug-size molecules include, but are not limited to, drugs, agrochemicals, aromatic substances, or fragrances.

[0191] One embodiment of the method of the present invention is to test the small molecule set in the biological macromolecular crystal system in order to separate the small molecules that interact with the corresponding biological macromolecules from the small molecule set that does not interact with the corresponding biological macromolecules.

[0192] Typically, in the arrays and methods of this invention, small molecules are dissolved in OS.

[0193] Definition of organic solvents (os)

[0194] A solvent is a substance that can dissolve other substances. The term organic solvent (OS) refers to any low-reactivity proton or aprotic carbon-based solvent capable of dissolving or dispersing one or more other substances. Carbon-based means that these solvents contain at least one carbon atom. In addition, they most usually contain at least one hydrogen atom. The terms "liquid carbohydrate" and "carbon-based solvent" have the same meaning and are used interchangeably.

[0195] Examples of suitable organic solvents are straight-chain or branched monoaliphatic alcohols containing 1 to 7 carbon atoms, such as ethanol or isopropanol; dialiphatic alcohols containing 2 to 7 carbon atoms, such as hexanediol; aliphatic diol monoalkyl ethers containing a total of 3 to 6 carbon atoms, such as ethylene glycol monomethyl ether, ethylene glycol ethyl ether, and ethylene glycol butyl ether; or dialkyl ketones containing a total of 3 to 5 carbon atoms, such as acetone. Other solvents that can be used are benzyl alcohol or phenethyl alcohol. Preferred solvents are ethanol, hexanediol, ethylene glycol monomethyl ether, and acetone. Solvent mixtures may also be used.

[0196] Organic solvents may be selected from a preferred group including, but not limited to, the following: methyl sulfoxide (dimethyl sulfoxide (DMSO)) and its derivatives, especially phosphorus derivatives; ethyl acetate and its derivatives; methanol; ethanol; propan-1-ol; propan-2-ol; N,N-dimethylformamide; oxacyclopentane (tetrahydrofuran); ethoxyethane (diethyl ether); crown ethers; 1,4-dimethylformamide; and others. Alkane (di) Alkane), furan and other solvents suitable for dissolving small molecules used in protein crystal screening.

[0197] According to the present invention, mixtures of individual organic solvents selected from the above-mentioned preferred organic solvent list can be used. Therefore, the term "organic solvent" according to the present invention can refer to a mixture of organic solvents. According to the present invention, mixtures of individual organic solvents selected from the above-mentioned preferred organic solvent list can be used in the array.

[0198] In a preferred embodiment, the small molecules are dissolved in OS.

[0199] Definition of compatible solute (permeation protectant, penetrant, or cryoprotectant): Compatible solutes (CS), osmotic protectants, or permeants include organic compounds of natural or synthetic origin that prevent protein precipitation or denaturation under stress conditions and maintain the spatial organization / conformational stability and solubility of peptides, proteins, and RNA. Such stress conditions include temperature changes (including cryogenic conditions), changes in osmotic stress, the presence of protein-denaturing compounds such as urea, drought, radiation, free radicals, poisons, and pressure. Naturally occurring compatible solutes have high solubility in water and low toxicity to organisms even at high concentrations, thus acting as permeants and helping organisms withstand extreme osmotic stress. Compatible solutes can be selected from methylamine, polyhydroxy compounds (including polymers, monosaccharides, disaccharides, trisaccharides, cyclic polyols), amino acids, and others.

[0200] In proteins and RNA molecules, compatible solutes have been shown to stabilize macromolecular conformations, particularly by inducing different conformations.

[0201] In a preferred embodiment, the compatible solute is selected from methylamine, polyhydroxy compounds (including polymers, monosaccharides, disaccharides, trisaccharides, cyclic polyols), and amino acids. In a most preferred embodiment, cs is polyethylene oxide, 2-methylpentane-2,4-diol (MPD), ethylene-1,2-diol (ethylene glycol, EG), and / or propylene-1,2,3-triol (glycerol) with a molecular weight of 400 Da (PEG400) or 3350 Da (PEG3350).

[0202] In some embodiments, cs is a liquid and is added to the soaking solution by volume Vos. Examples of liquid cs are polyoxyethylene (PEG400) with a molecular weight of 400 Da, propylene-1,2,3-triol (glycerol), ethylene-1,2-diol (ethylene glycol), and 2-methylpentane-2,4-diol (MPD).

[0203] In some embodiments, cs is a solid and is added to an immersion solution dissolved in water, where Vos refers to the volume of the corresponding cs dissolved in water. Examples of solid cs are N,N-dimethylamine oxide (trimethylamine N-oxide or TMAO), 2-trimethylammonium acetate (trimethylglycine or betaine), 2-(methylamino)acetic acid (N-methylglycine or sarcosine), 2-aminoethanesulfonic acid (taurine), 2-aminoethanesulfinic acid (hypoturic acid), and (2S)-pyrrolidine-2-carboxylic acid (proline).

[0204] In an additional specific embodiment of the invention, the preferred methylamine group includes N,N-dimethylmethylamine oxide (trimethylamine N-oxide or TMAO), 2-trimethylammonium acetate (trimethylglycine or betaine), 2-(methylamino)acetic acid (N-methylglycine or sarcosine), 2-hydroxyethyl(trimethyl)ammonium (choline), 2-[formamidinyl(methyl)amino]acetic acid (creatine), L-α-glycerophosphorylcholine, (R)-(3-carboxy-2-hydroxypropyl)-N,N,N-trimethylammonium hydroxide (carnitine), and corresponding stereoisomers and derivatives.

[0205] In another additional specific embodiment of the invention, the preferred amino acid group includes 2-aminoethanesulfonic acid (taurine), 2-aminoethanesulfinic acid (hypotaurine), (2S)-pyrrolidine-2-carboxylic acid (proline), 2-aminoacetic acid (glycine), (6S)-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (ectoine), (5S,6S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (hydroxyectoine), 4-aminobutyric acid (γ-aminobutyric acid), and (2S)-2-aminopentanediol. Acids (glutamic acid), β-hydroxy-γ-N-trimethylaminobutyric acid, (2S)-2-amino-3-methylbutyric acid (valine), (2S,3S)-2-amino-3-methylvaleric acid (isoleucine), (2S)-2-aminosuccinic acid (aspartic acid), (2S)-2-aminopropionic acid (alanine), 2-(methylamino)ethanesulfonic acid (N-methyltaurine), (2S)-2-[[(1R)-1-carboxyethyl]amino]-5-(diaminomethyleneamino)valeric acid (Octopin), and their corresponding stereoisomers and derivatives.

[0206] In an additional specific embodiment of the invention, the preferred group of polyhydroxy compounds includes polyoxyethylene, monosaccharides, disaccharides, trisaccharides, cyclic polyols, and derivatives of the aforementioned molecules.

[0207] In an additional specific embodiment of the invention, the preferred polyoxyethylene group includes polyhydroxy compounds of different molecular weights (e.g., polyethylene glycol 400), propylene-1,2,3-triol (glycerol), ethylene-1,2-diol (ethylene glycol), 2-methylpentane-2,4-diol (2-methylpentane-2,4-diol, MPD), (3R,4S,5S,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxacyclohexane-3,4,5-triol (1-glucosylglycerol), and PEG 3350 (also known as 3350 or polyethylene glycol 3350). In a preferred embodiment, the molecular weight of PEG is in the range of 200-20000 g / mol, including but not limited to polyoxyethylene (200 g / mol, 300 g / mol, 400 g / mol, 550 g / mol, 600 g / mol, 1000 g / mol, 1500 g / mol, 2000 g / mol, 3350 g / mol, 4000 g / mol, 5000 g / mol, 6000 g / mol, 8000 g / mol, 10000 g / mol, 20000 g / mol).

[0208] In a preferred embodiment, the compatible solute is a mixture of at least two polyoxyethylenes, preferably PEG400 and 3350.

[0209] In an additional specific embodiment of the invention, the preferred monosaccharide group includes (preferably: (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (glucose), (3S,4R,5R)-1,3,4,5,6-pentahydroxyhexane-2-one (fructose)), and (2S,3R,4S,5R,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxacyclohexyl-3,4,5-triol (isofloridoglycoside). In an additional specific embodiment of the invention, the preferred disaccharide group includes (β-D-fructofuranosyl-α-D-glucopyranoside (sucrose), (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxecyclohex-2-yl]oxyoxecyclohex-3,4,5-triol (trehalose)). In an additional specific embodiment of the invention, the preferred trisaccharide group comprises (preferably (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxetane-2-yl]oxo-6-[[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxetane-2-yl]oxetane-3,4,5-triol (raffinose)), and sugar alcohols (preferably (2R,3R,4R,5R)-hexyl-1,2,3,4,5,6-hexane (mannitol), (2S ... ,2,3,4,5,6-hexanediol (sorbitol), (1R,2S,3r,4R,5S,6s)-cyclohexyl-1,2,3,4,5,6-hexanediol (inositol), (2R,3R,4S)-pentan-1,2,3,4,5-pentanol (xylitol), (2R,3s,4S)-pentan-1,2,3,4,5-pentanol (ardentitol), (2R,3S)-butan-1,2,3,4-tetraol (erythritol), (2R,4R)-pentan-1,2,3,4,5-pentanol (arabinitol), (2R,3S,4R,5S)-hexyl-1,2,3,4,5,6-hexanediol (galactitol) In an additional specific embodiment of the invention, the preferred cyclic polyol group includes (preferably (1S,2S,4S,5R)-6-methoxycyclohexyl-1,2,3,4,5-pentaol (pine alcohol)). In an additional specific embodiment of the invention, the preferred derivatives of the aforementioned molecules include mannose derivatives such as fiorin ((2R)-O2-(β-D-mannopyranosyl)glyceric acid) and fiorin-A (mannosylglyceramide), inositol derivatives (preferably diinositol-1,1'-phosphate), glycerol derivatives (preferably cyclic 2,3-diphosphoglycerate, α-diglycerol phosphate), and polymers of the aforementioned molecules, such as starch, fructan, cellulose, and corresponding stereoisomers and derivatives.

[0210] In another specific embodiment of the invention, other preferred molecular groups include urea and its derivatives, 1-methyl-2-pyridinecarboxylic acid (lobster muscle), methyl sulfonate solutes such as 3-dimethylsulfonylpropyl ester (dimethylsulfonyl propyl ester) and methyl sulfoxide methane such as dimethyl sulfoxide, as well as other compounds having the characteristics of compatible solutes as defined, and corresponding stereoisomers and derivatives.

[0211] Definition of crystallization solution (crs)

[0212] A crystallization solution (crs) is any solution used at any point in time during any standard crystallization process to prepare crystals, particularly for crystal nucleation, growth, and / or storage under conditions suitable for maintaining crystal integrity for any further use. As an example, the crs of this invention can be defined according to various terms known in the art, such as mother liquor, nucleation medium, crystal growth medium, reservoir solution, and storage solution. Specifically, a crystallization solution includes mother liquor and other solutions such as reservoir solutions at any stage during the crystallization process.

[0213] In a preferred embodiment, the crystallization solution is obtained from the crystallization process of biomacromolecules and typically includes a buffer, precipitant, and additives to maintain pH. In this invention, crs refers to any solution used to prepare macromolecular crystals using any standard crystallization method known in the art. Ducruix, A. and Giegé, R. (eds.) *Crystallization of Nucleic Acids and Proteins* (Oxford University Press, Oxford, 1999); 1. Gavira, JA. *Current trends in protein crystallization*. Archives of Biochemistry and Biophysics 602, 3-11 (2016). 1. McPherson, A. and Gavira, JA. *Introduction to protein crystallization*. Acta Crystallographica Section FStructural Biology Communications F70, 2-20 (2014).

[0214] In other embodiments, the crystallization solution also refers to any solution that is not directly derived from the crystallization process, crystal nucleation, and growth medium. The crystallization solution also refers to a freshly prepared solution used to replace the crystal nucleation and growth medium. In particular, it refers to any solution used to store crystals in a suitable environment to maintain the crystal structure or integrity.

[0215] Therefore, crystallization solution (crs) refers to both 1) crystallization solution obtained from the crystallization process of biological macromolecules and 2) newly prepared organic or inorganic monovalent, divalent or trivalent salt solutions, such as chloride salts, preferably NaCl, MgCl2 or KCl.

[0216] In a later embodiment, the crystallization solution (crs) is also called an equivalent salt solution and refers to any solution that is not directly obtained from the crystallization process, crystal nucleation, and growth medium. The crystallization solution also refers to a newly prepared solution used to replace the crystal nucleation and growth medium.

[0217] CRS can also refer to a newly prepared solution that is not extracted from the crystallization process and the solution used to grow the crystal. In this embodiment, the newly prepared CRS typically consists of an aqueous solution of salt and / or precipitate, which is not the original CRS but still plays an equivalent role in maintaining net osmotic pressure, protein solubility, dielectric constant, and protein conformation in the soaking solution, and thus in maintaining crystal integrity. Typically, freshly prepared aqueous solutions consist of organic or inorganic monovalent, divalent, or trivalent salts, particularly sodium chloride, potassium chloride, diammonium sulfate (ammonium sulfate), sodium acetate, sodium citrate (such as sodium dihydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (monosodium citrate), disodium hydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (disodium citrate), trisodium 2-hydroxypropyl-1,2,3-tricarboxylate (trisodium citrate), dilithium sulfate, lithium chloride, lithium acetate, lithium formate, lithium nitrate, magnesium diacetate, sodium nitrate, sodium formate, and amphoteric salts, as well as precipitants such as polyoxyethylene, especially PEG3350, PEG4000, PEG8000, and others, such as MPD.

[0218] Advantageously, the freshly prepared CRS makes the process independent of the original CRS. Another advantage is that the same soaking solution can be used to soak different biomolecules, especially different protein crystals. Preferably, when the salt solution of the freshly prepared CRS is used as the soaking solution, the CRS is a mixture of PEGs, more preferably PEG400 and PEG3350.

[0219] Mother liquor is any solution containing concentrated proteins, peptides, or RNA used for crystallization. It may also contain buffers, precipitants, and additives to maintain pH. Reservoir solution is any solution containing buffers, precipitants, and additives to maintain pH, but typically does not contain proteins. In some vapor diffusion techniques, it is used to drive proteins in the mother liquor into supersaturation. Supersaturation is a non-equilibrium state where the amount of protein, peptide, or RNA present exceeds the solubility limit. Equilibrium is restored during crystallization. Supersaturated solutions are created by altering the properties of unsaturated solutions to reduce the medium's ability to dissolve large molecules. Techniques used to achieve supersaturation include batch crystallization, vial batching, sub-oil micro-batch processing, controlled evaporation, batch dialysis, concentration dialysis, microdialysis, free interface diffusion, capillary back diffusion, liquid bridges, vapor diffusion on a plate (sitting drop), hanging drop vapor diffusion, sequential extraction, pH-induced crystallization, temperature-induced crystallization, crystallization by adding effectors, and in vivo crystallization. Since the composition of the mother liquor and the reservoir solution changes during the process of the aforementioned techniques (e.g., through dialysis or vapor diffusion), the terms mother liquor and reservoir solution compiled in the term crystallization solution apply to the corresponding solutions at any point in time during the crystallization process.

[0220] The initial concentration of the crystallization solution can be a multiple of the original concentration of the crystallization solution used in the crystallization process. This multiple can be an integer multiple, such as 3 times, or a fractional multiple, such as 3.5 times or 0.8 times. In a preferred embodiment, the crystallization solution is used at one time the concentration of the original crystallization solution when it is used in the crystallization process. This means that the initial concentration of the crystallization solution used according to the invention is the same as the concentration of the crystallization solution used in the crystallization process.

[0221] Definition of precipitant

[0222] A precipitant is a chemical reagent that mediates the transformation of dissolved substances, such as biomolecules (e.g., proteins or RNA molecules), into insoluble solids. Precipitation occurs when the concentration of the substance to be precipitated exceeds its solubility. In crystallography, precipitants are used to form solids of biomolecules, but often, if unsuccessful, result in the formation of amorphous precipitates.

[0223] Definition of osmotic net pressure conditions

[0224] The transfer of protein crystals from their original crystallization environment to the immersion environment affects the composition of the solution. Multiple redistributions occur along a short-term concentration gradient on the crystal surface. If the concentration of a corresponding component in the immersion environment is higher than its concentration in the crystal's solvent channels, the component flows from the immersion environment into the crystal. Conversely, if the concentration of the component in the crystal's solvent channels is higher than its concentration in the immersion environment, the component flows out of the crystal's solvent channels into the immersion environment. These concentration differences lead to redistribution along the concentration gradient, generating osmotic pressure. This osmotic pressure, when combined, forms net osmotic pressure, which can cause compression or expansion of the crystal. In both cases, the crystal will fracture. To maintain the crystal under conditions suitable for crystallographic analysis after immersion, the net osmotic pressure at the crystal surface is crucial. Not exceeding Pa, to prevent crystal damage: It is an empirical threshold that depends on the individual protein of interest and most likely on its packaging, and defines the cutoff value for tolerable net osmotic pressure.

[0225] Definition of protein solubility conditions

[0226] The transfer of protein crystals from their original crystallization environment to an immersion environment introduces changes in water activity, salt activity, and additive activity. This affects the solubility (S) of the natural protein; if the solubility of biomolecules in the immersion environment is sufficiently increased compared to the original crystallization solution, crystal dissolution will occur. Therefore, to maintain the integrity of the crystals during the transfer from the crystallization environment to the immersion environment, the immersion environment must be adjusted to ensure that the solubility (S) is sufficient.

[0227] For the corresponding crystal, the following is given:

[0228] Where SS represents the solubility of the crystal in the soaking solution. S CS eq (This represents the solubility of the crystal in the original crystallization solution when the crystallization solution and the crystal are in equilibrium.) It is the free energy of a crystal when it is transferred from its original crystallization environment to the soaking environment. In order to prevent the crystal from dissolving, the free energy must be unfavorable.

[0229] Definition of protein conformation conditions

[0230] When biomacromolecule crystals are transferred from their original crystallization solution to an immersion environment, new components not present in the crystallization solution are introduced into the crystals using the immersion solution. This specifically involves the introduction of organic solvents, compatible solutes, and finally, small molecules from the screening library. Such components can cause denaturation of individual biomacromolecules, meaning a disturbance, partial or complete loss of the macromolecule's native conformation. Loss of native conformation can lead to a loss of the integrity of the macromolecular crystal, subsequently resulting in the dissolution of its (partially) denatured form. Therefore, the effect of the denaturing component must be compensated for by components that preserve the structure. Organic solvents are typically used at concentrations suitable for screening small molecules to exert a denaturing effect on biomacromolecules. Compatible solutes are natural compounds that can compensate for this effect and preserve the native conformation of biomacromolecules. These molecules originate from organisms that, in evolutionary history, required protectants to withstand high-stress conditions such as drought, cold, high salt, or urea concentrations. Their general function is to preferentially hydrate the macromolecules. Preferential hydration means that proteins prefer to interact with water rather than compatible solutes, which are then excluded from the direct protein surface. In contrast, denaturing agents preferentially interact with the biomacromolecules themselves, especially with the protein backbone. This results in the exclusion of water from the immediate surroundings of the macromolecule and the enrichment of the denaturing agent. The preferential interaction of biomacromolecules with water exerts a hydrophobic effect, leading to the folding reaction of the corresponding biomacromolecule, thereby producing its native conformation. The replacement of water by the denaturing agent produces the opposite effect. Therefore, the effect of the denaturing agent can be compensated by using a suitable compatible solute that enhances the hydration state of the biomacromolecule. This stabilizes the native conformation of the individual biomacromolecules in the crystal lattice, thus stabilizing the integrity of the crystal lattice. Since the interactions of individual biomacromolecules with organic solvents and compatible solutes differ, suitable combinations of both must be identified. The technique proposed here achieves this condition by means of a systematic evaluation of suitable combinations. This condition can be further divided into two sub-conditions: a. Precipitation conditions It is the free energy by which the native protein dissolved in the original crystallization solution (where it is in equilibrium with the protein incorporated into the crystal after crystallization) is transferred to the immersion solution. It must be zero or necessarily unfavorable because the immersion solution cannot increase the solubility of the protein. In the case of proteins, for Two contributing aspects are the contribution to the free energy generated by the amino acid side chains exposed on the surface of the native protein during this transfer. This contribution is generated by the solvent-accessible portion of the natural protein backbone.

[0231] b. Stability conditions It refers to the change in free energy when unfolding in an immersion environment. This refers to the change in free energy during unfolding in a crystalline environment. If the difference is greater than zero, the native conformation in the immersion solution is favorable.

[0232] Definition of dielectric constant condition

[0233] The dielectric constant is a unit of measurement for the polarizability of a dielectric. When protein crystals are transferred from the original crystallization solution to the immersion environment, the dielectric constant of the solution may change due to the introduction of organic solvents, compatible solutes, and small molecules used for small molecule screening. Because of the multiple redistributions of these components, the dielectric constant within the crystal may differ after crystal transfer and reequilibrium. This means that the polarization of the original crystallization solution or the immersion solution in response to an electric field may be greater or less than that of the other. Since the crystallization process requires the use of ions in the crystallization solution to screen the charges of macromolecules and thus retains these ions after crystal formation, the dielectric constant... Changes exceeding y will cause the crystal's electrostatic setting to be lost.

[0234]

[0235] y is an individual threshold that depends on the individual type of the macromolecular crystal and most likely on its packaging, and defines the tolerable dielectric constant. The cutoff value for the change.

[0236] Definition of methods for controlling biomolecule crystals and selecting appropriate soaking solutions

[0237] In this invention, the crystal control step can be performed by any method available to a person skilled in the art to assess the presence and / or aspects of the crystal and its suitability for further analysis (such as X-ray diffraction analysis). The crystal control step refers to assessing the survival rate of at least one biomacromolecule crystal in a specific immersion solution in terms of its presence and measuring the effect of the immersion solution on the crystal's integrity / stability. Preferred methods include X-ray diffraction, or neutron diffraction at an internal source or synchrotron, and visual analysis of the crystal using UV / VIS and IR spectroscopy.

[0238] In a specific embodiment of the invention, the crystal is controlled using conventional techniques such as standard X-ray analysis and / or by visual control under a microscope, and then the immersion solution is selected. In a preferred embodiment, X-ray analysis is performed after visual control.

[0239] In a preferred embodiment, more than one crystal is controlled. In a most preferred embodiment, crystals derived from at least two different biomacromolecules are controlled in the same solution composition.

[0240] In a fully automated setup, a robot grasps the experimental plate, artificial intelligence identifies the crystals in the holes (or discards the corresponding holes if the crystals are dissolved or in poor condition), and automatically transfers the selected crystals to an X-ray beam. Preferably, the final decision is made via X-ray evaluation. Additionally, visual inspection can be used to identify crystals in the holes of the experimental plate that are worthy of X-ray analysis. In a preferred embodiment, all types of crystals or crystal residues are controlled by X-ray analysis, as sometimes poorly shaped crystals can diffract well. In another preferred embodiment, only empty holes where the crystals are completely dissolved or broken are discarded.

[0241] Visual inspection can be performed under a microscope or using UV / VIS spectroscopy or IR spectroscopy to identify surviving crystals that can be analyzed by X-ray.

[0242] Visual inspection is preferred to observe how the appearance of the crystals changes over time. This can be used to estimate the maximum immersion time.

[0243] Visual data can be collected to correlate penetration conditions in the array with rupture behavior and other factors.

[0244] In a preferred embodiment, the composition of the immersion solution can be first selected after visual analysis of the crystal. Immersion solution compositions that result in complete dissolution or severe damage to the crystal, such as cracking, are discarded. The first set of immersion solutions selected after visual control can then be subjected to X-ray analysis. This involves crystals from the immersion solution compositions showing no signs of damage under visual analysis (i.e., sharp edges and smooth crystal faces, no cracks, visible color under polarized light) or moderate signs of damage, without affecting the general possibility of transferring the corresponding crystal and performing X-ray diffraction or neutron diffraction experiments on it. With X-ray diffraction or neutron diffraction analysis, a technician can set all parameters using only common knowledge.

[0245] The method of controlling crystals and selecting a suitable immersion solution according to the present invention does not require any specific knowledge beyond the common knowledge of experts in protein crystallization. Parameters of the control method, such as maximum resolution, low mosaicism, intact crystal structure, electron density quality, absence of twins due to unwanted ice crystals, and absence of diffraction signals, are set to allow for standard control of crystal integrity, after which a suitable immersion solution is selected.

[0246] For visual control and X-ray control, it is possible to... Figures 6 to 7 and Figures 10 to 11 The template shown makes it easy to report parameters.

[0247] For visual inspection / control, the observer notices the presence of even the smallest fractures or cracks. If none are present or only minimal dissolution is observed, the sharp crystal edges and color in polarized light are assessed. Specifically, the selection criteria for visual inspection can be based on the presence or absence of cracks in the crystal, or the type of crack. Cracks are typically classified (longitudinal cracks, transverse cracks, and cross-cutting cracks). The appearance of the crystal surface (e.g., smooth, rough) or the appearance of its edges (e.g., sharp, rounded) and their characteristics in polarized light (strong color, colored, colorless) are standard criteria for assessing the impact of the immersion solution composition on the crystal and selecting the immersion solution that best preserves the crystal's integrity. In most cases, not all crystals can withstand all conditions. Some crystals dissolve completely or are damaged over time, while others retain their integrity entirely.

[0248] X-ray examinations can be performed using commercially available X-ray sources as well as on a synchrotron. Commercially available X-ray sources are much slower in terms of data acquisition. For this reason, as a precaution, at least two images are typically taken at 0° and 90° at different crystal orientations. These images are based on sufficient data to assess their quality after the crystal has undergone appropriate immersion in the solution, including resolution and mosaicism, artifact-like data arising from unwanted ice crystals in protein crystals, and the sometimes known phenomenon known as "twins."

[0249] For X-ray examination, it is preferable to remove the crystal from the cryogenic vial or any other storage container and fix it in a so-called cryogenic stream, such as a protein crystal, on an X-ray or neutron diffractometer to maintain a low temperature around it. In the case of room-temperature X-ray studies, a cryogenic stream is not required. Technicians will typically define standard guidelines commonly used by experts in the field, such as: the highest resolution should be set less than 2.8 Å, more preferably 2.5 Å; low mosaicism should generally be less than 0.8° (depending on the detector); no or minimal ice rings; high-quality electron density; and minimal twinning.

[0250] Data collected during the crystallography control process is analyzed to select the immersion solution. The solution chosen improves crystal quality or has little or no adverse effect on the crystal, according to quality standards. Selection criteria can be determined by crystallographers using common knowledge.

[0251] Data collection is much faster on synchrotrons. In this case, the complete dataset is collected and evaluated, as the emergence of the so-called "electron density" becomes an additional verification criterion for experienced crystallographers to judge its suitability.

[0252] In one specific embodiment of the invention, the biomolecular crystals are controlled at least once, particularly by inspection using a combination of X-ray and preferably visual inspection, over a time range of 0 hours to several weeks, preferably after 0 hours, 1 hour, and 24 hours. In diffraction experiments, repeated inspections over time are performed to determine the maximum amount of immersion time required to maintain suitable diffraction quality.

[0253] Based on data collected through X-ray, neutron diffraction, and / or visual analysis, decisions are made regarding which soaking solution to use for further applications, particularly for small molecule fragment screening, molecular probe analysis, screening for more drug-sized small molecules, and validating HTS hits or biophysical screening hits.

[0254] In contrast to conventional soaking solution optimization, which yields a single soaking condition, the method of this invention typically provides a range of soaking conditions. From this range, the experimenter can further select the optimal soaking solution to address any adverse effects on the crystals that may arise from, for example, the corresponding small molecule fragments utilized or pH changes resulting from their utilization.

[0255] In a preferred embodiment, another control method consisting of stress testing the crystal can be used. This method is particularly useful for evaluating the tolerance of biomacromolecule crystals to changes introduced by the addition of small molecules.

[0256] Typically, stress testing is performed as an additional step following any of the methods described above for controlling crystal selection. Stress testing is often used to fine-tune the selection of an initial range of immersion solutions, first controlled and selected using X-ray or neutron diffraction at an internal source or synchrotron, and then visually analyzing the crystal.

[0257] For stress testing, the volume and composition of the selected solution remain constant, but the solution contains small molecules called stress probes. When using high concentrations of these small molecules, such stress conditions occur after their introduction, resulting in pH changes or changes in osmotic conditions. Stress testing typically consists of adding, for example, a hydrochloric acid fragment / probe for stress testing and fragments dissolved in organic solvents at varying concentrations in a previously selected soaking solution.

[0258] Definition of aqueous solution

[0259] The term "aqueous solution" describes a system containing water, while the term "solution" is a system in which one substance (solvent) dissolves another substance (solute). Therefore, an aqueous solution is a solution of water, the solvent for another substance, namely the solute. Polar or hydrophilic solutes are more readily soluble in water, such as salts, while hydrophobic or nonpolar solutes are soluble only in trace amounts (oils). However, trace amounts of nonpolar substances can have a significant impact on the properties of aqueous solutions.

[0260] The following embodiments are the subject of this invention: 1. A method for selecting an immersion solution suitable for immersing biomacromolecules in crystalline form, wherein the solution comprises an organic solvent (OS), a compatible solute (CS), a crystallization solution (CRS), and / or water (W), the method comprising the following steps 1) The soaking solution is prepared by mixing OS, CS, CRS, and W. 2) Transfer at least one crystal of the biomacromolecule to the solution prepared in step 1), or transfer the solution prepared in step 1) to a compartment containing at least one crystal of the biomacromolecule. 3) Control the crystal, 4) Select a suitable soaking solution for soaking the crystals.

[0261] 2. A method for selecting an immersion solution for immersing a biomacromolecule in crystalline form, wherein the immersion solution comprises an organic solvent (OS), a compatible solute (CS), a crystallization solution (CRS), and / or water (W); the method comprising the following steps

[0262] 1) Prepare a first series of 1n to xn individual soaking solutions by mixing os, cs, crs, and w, wherein: a) x is at least 2, b) Each soaking solution in the first series contains the same organic solvent (os), the same compatible solute (cs), and the same crystallization solution (crs). c) Vos and Vcs are the same in every solution. d) Vcrs and Vw vary in each solution. In solution 1n, Vcrs is the minimum Vcrs (Vcrsmin), and Vw is the maximum Vw (Vwmax). In solution xn, Vw is the minimum Vw (Vwmin), and Vcrs is the maximum Vcrs (Vcrsmax). When x>2, in any additional solution between 1n and xn, Vcrs varies between Vcrsmin and Vcrsmax, and conversely, Vw varies between Vwmin and Vwmax; 2) Transfer at least one crystal of the biomacromolecule from each compartment to x compartments, each compartment containing one of the solutions prepared in step 1); or transfer each solution prepared in step 1) to x compartments, each compartment containing at least one crystal of the biomacromolecule from its crystallization solution. 3) Control the crystal in each compartment; 4) Select a suitable soaking solution for soaking the crystals.

[0263] 3. The method for selecting the composition of the soaking solution according to any one of embodiments 1 to 2, the method further comprising the following steps:

[0264] 1) Prepare m series of 1n to yn individually soaked solutions by mixing os, cs, crs, and w, wherein m is at least 1, and wherein in each of the m series: a) y is at least 2, b) OS and CRS are the same as in the first series. c) Vos and Vcs are the same as in the first series. d) The cs is changed and is different from the cs of the first series. In each individual solution of each of the m series: e) Vos and Vcs remain unchanged. f) Vcrs and Vw vary in each solution. In solution 1n, Vcrs is the minimum Vcrs (Vcrsmin), and Vw is the maximum Vw (Vwmax). In solution yn, Vw is the minimum Vw (Vwmin), and Vcrs is the maximum Vcrs (Vcrsmax). When y>2, in any additional solution between 1n and yn, Vcrs varies between Vcrsmin and Vcrsmax, and conversely, Vw varies between Vwmin and Vwmax; 2) Transfer at least one crystal of the biomacromolecule from each compartment to y compartments, each compartment containing one of the solutions prepared in step 1); or transfer each solution prepared in step 1) to y compartments, each compartment containing at least one crystal of the biomacromolecule from its crystallization solution.

[0265] 4. The method for selecting the composition of the soaking solution according to any one of embodiments 1 to 3, wherein the crs is a salt solution different from the original crystallization solution used for nucleation, growth and / or storage of macromolecular crystals, wherein the salt of the crs is any salt that can be used for the crystallization of biological macromolecules.

[0266] 5. The method for selecting the composition of the soaking solution according to Embodiment 4, wherein the salt is NaCl, MgCl2 or KCl.

[0267] 6. The method for selecting the composition of the soaking solution according to Embodiment 4, wherein the salt is not a chloride (Cl) salt, and in particular not NaCl.

[0268] 7. The method for selecting the composition of the soaking solution according to any one of embodiments 1 to 6, wherein the percentage of the total volume (VT) of each solution in the first series is VT = Vcrs + Vw + Vos + Vcs = 100%.

[0269] 8. The method for selecting the composition of the soaking solution according to any one of embodiments 1 to 7, wherein the step of controlling the crystal is performed by X-ray diffraction, neutron diffraction, absorption or reflection spectroscopy in the ultraviolet and visible spectral regions, infrared spectroscopy and / or visual analysis.

[0270] 9. The method for selecting the composition of an immersion solution according to any one of embodiments 1 to 8, wherein the OS contains small molecules, including small fragments and small probes.

[0271] 10. A method for small molecule screening of biological macromolecular crystals, wherein the screening is performed in an immersion solution selected according to the method described in embodiment 9.

[0272] 11. An immersion solution, said immersion solution being obtained by means of selecting the composition of the immersion solution according to any one of embodiments 1 to 9.

[0273] 12. The soaking solution according to embodiment 11, wherein the Vos is at least 5%.

[0274] 13. Use of the soaking solution according to any one of embodiments 11 or 12 for small molecule screening of biological macromolecular crystals.

[0275] 14. An array arranged to perform the method according to any one of embodiments 1 to 13.

[0276] 15. An array containing a solution according to any one of embodiments 11 or 12.

[0277] The following embodiments are also the subject of this invention: 1. A method for selecting an immersion solution for immersing a biomacromolecule in crystalline form, wherein the immersion solution comprises an organic solvent (OS), a compatible solute (CS), a crystallization solution (CRS), and / or water (W); the method comprising the following steps 1) Prepare a first series of 1n to xn individual soaking solutions by mixing os, cs, crs, and w, wherein: a) x is at least 2, b) Each soaking solution in the first series contains the same organic solvent (os), the same compatible solute (cs), and the same crystallization solution (crs). c) In every solution, Vos and Vcs are the same, or, in every solution, Mos / Mcs is the same. d) Vcrs and Vw vary in each solution. In solution 1n, V crs For the minimum V crs (V) crs min), V w For the maximum V w (V) w max). In solution xn, V w For the minimum V w (V) w min), V crs For the maximum V crs (V) crs max). When x > 2, in any additional solution between 1n and xn, V crs In V crs min and V crs The values ​​of max and V vary, and conversely, V... w In V w min and V w Variations between max values; 2) Transfer at least one crystal of the biomacromolecule from each compartment to x compartments, each compartment containing one of the solutions prepared in step 1); or transfer each solution prepared in step 1) to x compartments, each compartment containing at least one crystal of the biomacromolecule from its crystallization solution. 3) Control the crystals in each compartment; 4) Select a suitable soaking solution for soaking the crystals; The compatible solute is selected from polyhydroxy compounds, amino acids, methylamine, and mixtures thereof. The organic solvent is selected from low-reactivity proton or aprotic liquid carbohydrates and mixtures thereof capable of dissolving small molecules, and the crystallizing solution crs is obtained from the crystallization process of the crystal or is equivalent to it in terms of net osmotic pressure, dielectric constant, solubility of the corresponding biomacromolecule, and effect on the structural conformation of the corresponding biomacromolecule, and contains pH and buffering substances, additives and precipitants.

[0278] 2. The method for selecting the composition of the soaking solution according to Embodiment 1, wherein the compatible solute is selected from polyhydroxy compounds, amino acids, methylamine, or mixtures thereof, wherein the polyhydroxy compounds are selected from polyoxyethylene, polyols, monosaccharides, disaccharides, trisaccharides, sugar alcohols, cyclic polyols, and derivatives of the aforementioned molecules, wherein the amino acids are selected from 2-aminoethanesulfonic acid (taurine), 2-aminoethanesulfinic acid (hypoturonic acid), (2S)-pyrrolidine-2-carboxylic acid (proline), 2-aminoacetic acid (glycine), (6S)-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (ectoin), (5S,6S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (hydroxyectoin), 4-aminobutyric acid (γ-aminobutyric acid), (2S)-2-aminoglutarate (glutamic acid), β-hydroxy-γ-N-trimethylaminobutyric acid, (2S)-2-amino-3-aminobutyric acid, and 4-aminobutyric acid (γ-aminobutyric acid), (2S)-2-aminoglutarate (glutamic acid), β-hydroxy-γ-N-trimethylaminobutyric acid, and (2S)-2-amino-3-aminobutyric acid. 2-Methylbutyric acid (valine), (2S,3S)-2-amino-3-methylvaleric acid (isoleucine), (2S)-2-aminosuccinic acid (aspartic acid), (2S)-2-aminopropionic acid (alanine), 2-(methylamino)ethanesulfonic acid (N-methyltaurine), (2S)-2-[[(1R)-1-carboxyethyl]amino]-5-(diaminomethyleneamino)valeric acid (Octopin), wherein the methylamine is selected from N,N - Dimethylamine oxide (trimethylamine N-oxide or TMAO), 2-trimethylammonium acetate (trimethylglycine or betaine), 2-(methylamino)acetic acid (N-methylglycine or sarcosine), 2-hydroxyethyl(trimethyl)ammonium (choline), 2-[formamidinyl(methyl)amino]acetic acid (creatine), L-α-glycerophosphorylcholine, (R)-(3-carboxy-2-hydroxypropyl)-N,N,N-trimethylammonium hydroxide (carnitine).

[0279] 3. The method for selecting the composition of the soaking solution according to Embodiment 1 or 2, wherein the compatible solute is selected from polyhydroxy compounds, amino acids, methylamine, or mixtures thereof, wherein the polyhydroxy compounds are selected from polyoxyethylene, polyols, monosaccharides, disaccharides, trisaccharides, sugar alcohols, cyclic polyols, and derivatives of the aforementioned molecules, wherein the polyoxyethylene group is selected from molecules with a molecular weight of 20-2,000,000. Monodisperse or polydisperse polyethylene oxides in the range of g / mol, including but not limited to, for example, polyethylene glycol 400 and polyethylene glycol 600, wherein the polyol is selected from propan-1,2,3-triol (glycerol), ethylene-1,2-diol (ethylene glycol), 2-methylpentan-2,4-diol (2-methylpentan-2,4-diol), (3R,4S,5S,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxacyclohexane-3,4,5-triol (1-glucosylglycerol), wherein the monosaccharide is selected from (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (glucose), (3S... (2S,3R,5R)-1,3,4,5,6-pentahydroxyhexane-2-one (fructose), (2S,3R,4S,5R,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxacyclohexyl-3,4,5-triol (isofloridoside), wherein the disaccharide group is selected from β-D-fructofuranosyl-α-D-glucopyranoside (sucrose), (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxacyclohexyl-2-yl]oxyoxacyclohexyl-3,4,5-triol (trehalose), wherein The trisaccharide group is selected from (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxetane-2-yl]oxo-6-[[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxetane-2-yl]oxetane]oxetane-3,4,5-triol (raffinose), wherein the sugar alcohol group includes (2R,3R,4R,5R)-hexyl-1,2,3,4,5,6-hexane (mannitol) and (2S,3R,4R,5R)-hexyl-1,2,3,4,5,6-hexane (amylose). The group of cyclic polyols includes (1R,2S,3r,4R,5S,6s)-cyclohexyl-1,2,3,4,5,6-hexane (inositol), (2R,3R,4S)-pentan-1,2,3,4,5-pentanol (xylitol), (2R,3s,4S)-pentan-1,2,3,4,5-pentanol (adamantol), (2R,3S)-butan-1,2,3,4-tetraol (erythritol), (2R,4R)-pentan-1,2,3,4,5-pentanol (arabinitol), and (2R,3S,4R,5S)-hexyl-1,2,3,4,5,6-hexane (galactitol), wherein the cyclic polyol group includes (1S,2S,4S,5R)-6-methoxycyclohexyl-1,2,3,4,5-pentaol (pineol) and (2S,3R,4S,5R,6R)-2-[(1,3-dihydroxypropyl-2-yl)oxy]-6-(hydroxymethyl)oxacyclohexyl-3,4,5-triol (floridoside), wherein the derivatives of the aforementioned molecules include mannose derivatives such as fiorin ((2R)-O2-(β-D-mannopyranosyl)glyceric acid) and fiorin-A (mannosylglyceramide), inositol derivatives (preferably diinositol-1,1'-phosphate), glycerol derivatives (preferably cyclo2,3-diphosphoglycerate, α-diglyceride), and polymers of the aforementioned molecules, such as starch, fructan, and cellulose.

[0280] 4. The method for selecting the composition of the soaking solution according to any one of embodiments 1 to 3, wherein the organic solvent is a straight-chain or branched monoaliphatic alcohol, a dialiphatic alcohol, a monoalkyl ether of an aliphatic diol, an ether of aliphatic monoaliphatic alcohol, a cyclic non-aromatic ether, a (cyclic)enol ether, a dialkyl ketone, benzyl alcohol, phenethyl alcohol, (alkylated) sulfoxide and corresponding phosphorus derivatives, esters derived from acetic acid, (alkylated) formamide or mixtures thereof.

[0281] 5. The method for selecting the composition of the soaking solution according to embodiments 1 to 4, wherein the crystallization solution CRS is obtained from the crystallization process of the crystal or is equivalent to it in terms of net osmotic pressure, dielectric constant, solubility of the corresponding biomacromolecule, and effect on the structural conformation of the corresponding biomacromolecule, and comprises pH and buffering substances, additives and precipitants or mixtures thereof, wherein the pH and buffering substances are selected from acetate buffer, TRIS buffer, HEPES buffer, dimethyl arsenate buffer, 1H-imidazole (imidazole) buffer and citrate buffer, wherein the precipitant is selected from volatile organic solvents, salts, polymers or non-volatile organic solvents, wherein the additives are selected from physiologically or biochemically relevant small molecules, chemical protectants, solubilizers and cleaners, compounds that reduce twinning, penetrants, cosolvents and structure-making agents, compounds that crosslink carboxyl and amino groups on protein surfaces, compounds that form electrostatic, covalent or hydrogen-bonded arrangements intended to stabilize biomacromolecule crystals, and substances or compounds that enhance nucleation and provide unique surfaces.

[0282] 6. The method for selecting the composition of the soaking solution according to embodiments 1 to 5, wherein the crystallization solution CRS is obtained from the crystallization process of the crystal or is equivalent to it in terms of net osmotic pressure, dielectric constant, solubility of the corresponding biomolecule, and effect on the structural conformation of the corresponding biomolecule, and comprises pH and buffer substances, additives and precipitants or mixtures thereof, wherein the precipitant is selected from volatile organic solvents, salts, polymers or non-volatile organic solvents, wherein the volatile organic solvent is selected from ethanol, propan-1-ol, propan-2-ol, 2-propanol, 1,4-diol, etc. Alkane (di) Alkane), propan-2-one (acetone), 2-methylpropan-1-ol (isobutanol), butan-1-ol (n-butanol), 2-methylpropan-2-ol (tert-butanol), acetonitrile, methyl sulfoxide (dimethyl sulfoxide), oxepane-2-one (γ-butyrolactone), 4-methyloxepane-2-one (β-butyrolactone), wherein the salt is selected from disodium 2,3-dihydroxysuccinate (sodium tartrate), (2R,3R)-2,3-dihydroxysuccinate (dipotassium tartrate), ammonium phosphate (ammonium phosphate), diammonium sulfate (ammonium sulfate), ammonium 2-hydroxypropan-1,2,3-tricarboxylate (ammonium citrate), sodium acetate (sodium acetate), ammonium acetate (ammonium acetate), lithium sulfate, lithium chloride, sodium chloride, potassium chloride, sodium formate (sodium formate), sodium and potassium monophosphates, sodium and potassium diphosphates. Salts and polyphosphates, sodium citrate (such as sodium citrate dihydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (monosodium citrate), disodium 2-hydroxypropyl-1,2,3-tricarboxylate (disodium citrate), trisodium 2-hydroxypropyl-1,2,3-tricarboxylate (trisodium citrate), magnesium dicarboxylate, magnesium dichloride, magnesium sulfate, calcium chloride, disodium succinate (sodium succinate), cadmium(II) sulfate (cadmium sulfate), disodium malonate (sodium malonate), magnesium diacetate (magnesium acetate), zinc diacetate (zinc acetate), calcium diacetate (calcium acetate), wherein the polymer is selected from polyethylene glycol (preferably PEG400, PEG1000, PEG3350, PEG4000, PEG6000, PEG8000, PEG20000), Jeffamine T, Jeffamine M, 2-methoxyethanol (polyethylene glycol monomethyl ester), 2-hydroxyethyl octadecanoate (polyethylene glycol monostearate), and polyenamine, wherein the non-volatile organic solvent is selected from 2-methyl-2,4-pentanediol, hexane-2,5-diol (2,5-hexanediol), propane-1,3-diol (1,3-propanediol), polyethylene glycol (e.g., 400), and Jeffamine (e.g., 400).

[0283] 7. A method for selecting the composition of an immersion solution according to embodiments 1 to 6, wherein the crystallization solution CRS is obtained from the crystallization process of the crystal or is equivalent to it in terms of net osmotic pressure, dielectric constant, solubility of the corresponding biomacromolecule, and effect on the conformation of the corresponding biomacromolecule, and comprises pH and buffering substances, additives and precipitants or mixtures thereof, wherein the additive group is selected from physiologically or biochemically relevant small molecules, chemical protectants, solubilizers and detergents, compounds that reduce twinning, penetrants, cosolvents and structure-forming agents, compounds that crosslink carboxyl and amino groups on protein surfaces, compounds that form electrostatic, covalent, or hydrogen-bonded arrangements aimed at stabilizing biomacromolecule crystals, and compounds that enhance nucleation and Provides a class of substances or compounds with unique surfaces, wherein the small molecules of protein physiological or biochemical relevance are selected from 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiophene[3,4-d]imidazol-4-yl]valerate biotin, [[(2R,3S,4R,5R)-5-(6-aminopurine-9-yl)-3,4-dihydroxyoxacyclopentan-2-yl]methoxy-hydroxyphosphoryl]phosphonate hydrophosphate (adenosine triphosphate), [(2R,3S,4R,5R)-5-(6-aminopurine-9-yl)-3,4-dihydroxyoxacyclopentan-2-yl]methylphosphonate hydrophosphate (adenosine diphosphate), [(2R,3S,4R,5R)-5-(6-aminopurine-9-yl)-3,4-dihydroxyoxacyclopentan-2-yl]methylphosphonate hydrophosphate (adenosine diphosphate), [(2R,3S,4R, 5R)-5-(6-aminopurine-9-yl)-3,4-dihydroxyoxacyclopentan-2-yl]methyl dihydrogen phosphate (adenosine monophosphate), 3-[18-(2-carboxyethyl)-7,12-bis(vinyl)-3,8,13,17-tetramethyl-21H,23H-porphyrin-2-yl]ferric propionate (Fe-protoporphyrin IX or heme b), wherein the chemical protectant is selected from 2-mercaptoethanol (β-mercaptoethanol or BME) and (2S,3S)-1,4-bis(mercapto)but-2,3-diol (dithiothreitol or DTT), 3-[bis(2-carboxyethyl)phosphonyl]propionate (TCEP), heavy metal ion scavengers such as 2-[2-[bis(carboxymethyl)amino]ethyl] [2-[2-[2-[2-[2-[bis(carboxymethyl)amino]ethoxy]ethoxy]ethyl-(carboxymethyl)amino]acetic acid (EGTA), compounds that prevent microbial infection such as sodium azide, phenol, 1,1,1-trichloro-2-methylprop-2-ol (chlorobutanol), wherein the solubilizer and detergent are selected from quaternary ammonium salts, sulfobetaine, dissociating agents such as urea, and surfactants and detergent molecules such as (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-octaoxyoxacyclohexane-3,4,5-triol (n-octyl-β-d-glucoside), octyl-polyoxyethylene (octyl-POE), N,N-Dimethyldodecane-1-amine oxide (lauryl dimethylamine oxide), (2R,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6R)-6-dodecyloxy-4,5-dihydroxy-2-(hydroxymethyl)oxetane-3-yl]oxo-6-(hydroxymethyl)oxetane-3,4,5-triol (dodecyl-β-D-maltodextrin), sodium 1,4-bis(2-ethylhexyloxy)-1,4-dioxobut-2-sulfonate (bis(2-ethylhexyl)sulfosuccinate), dodecyl ammonium chloride (dodecyl ammonium chloride), polyethoxylated fatty acids, wherein the compound intended to reduce twinning is selected from ethanol, methyl sulfoxide (dimethyl sulfoxide or DMSO), acetone (acetone), 1,4-di-di-, Alkane (di) alkyl, but-1-ol (butanol), 2-methylpentane-2,4-diol (2-methyl-2,4-pentanediol or MPD), wherein the penetrant, co-solvent and structure-forming agent are selected from (2R,3R,4S,5S,6R)-2-[(2S,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxetane-2-yl]oxo-6-(hydroxymethyl)oxetane-3,4,5- Triols (sucrose), (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxetane-2-yl]oxyoxetane-3,4,5-triols (trehalose) and other sugars, (2S)-pyrrolidine-2-carboxylic acid (proline), N,N-dimethylamine oxide (trimethylamine N-oxide or TM) AO), 2-aminoacetic acid (glycine), 2-(trimethylammonium)acetate (betaine), 2-aminoethanesulfonic acid (taurine), 2-(methylamino)acetic acid (sarcosine), wherein the category of compounds that crosslink carboxyl and amino groups on the protein surface is selected from molecules or aliphatic moieties containing diamino or dicarboxylic acids of different lengths carrying charged groups, wherein the category of compounds that form electrostatic, covalent, or hydrogen-bonded arrangements aimed at stabilizing the crystal through intermolecular crosslinking between proteins in the crystal is glutaraldehyde (glutaraldehyde), wherein the category of substances or compounds used to enhance nucleation and provide a unique surface is selected from PEG, Jeffamine, gels, such as gels used for cubic lipid phase crystallization and surfaces that promote epitaxial and heterogeneous nucleation, commercially available nucleating agents such as Crystallophore No. 1, Naomi nucleating agent, JBS Magical Triangle, Anderson-Evans polyoxotungstate (Na6[TeW6O24]·22 H2O) or buffer solution components that affect the solubility and aggregation properties of macromolecules, thus influencing their nucleation and growth properties, such as 2-aminoacetic acid (glycine), 3-[4-(3-sulfopropyl)piperazin-1-yl]prop-1-sulfonic acid (PIPPS), 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)prop-1,3-diol (Bis-Tris), 2-morpholino-4-ylethanesulfonic acid (MES), 2-[(2-amino-2- [Oxyethyl)-(carboxymethyl)amino]acetic acid (ADA), 2-[3-[[1,3-dihydroxy-2-(hydroxymethyl)prop-2-yl]amino]propylamino]-2-(hydroxymethyl)prop-1,3-diol (Bis-Tris propane), 3-morpholino-4-ylprop-1-sulfonic acid (MOPS), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-amino-2-(hydroxymethyl)prop-1,3-3-Diol (TRIS), 3-[4-(2-hydroxyethyl)piperazin-1-yl]prop-1-sulfonic acid (EPPS), 1H-imidazolium (imidazolium), 2-[bis(2-hydroxyethyl)amino]acetic acid (dihydroxyethylglycine), 2-(cyclohexylamino)ethanesulfonic acid (CHES), 3-(cyclohexylamino)prop-1-sulfonic acid (CAPS), (2R,3S,4S,5R)-2-(hydroxymethyl)-6-octaoxyoxacyclohexyl-3,4,5-triol (octyl) (2R,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6R)-6-dodecyloxy-4,5-dihydroxy-2-(hydroxymethyl)oxetane-3-yl]oxo-6-(hydroxymethyl)oxetane-3,4,5-triol (dodecyl maltodextrin), (2S,3S)-1,4-bis(mercapto)but-2,3-diol (DTT), and 3-[bis(2-carboxyethyl)phosphono]propionic acid (TCEP).

[0284] 8. The method for selecting the composition of an immersion solution according to any one of embodiments 1 to 7, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is a polyhydroxy compound a or a mixture thereof, and the organic solvent (os) is a low-reactivity proton or aprotic liquid carbohydrate or a mixture thereof capable of dissolving small molecules. More preferably, the compatible solute (cs) is polyethylene oxide, and the organic solvent (os) is selected from (alkylated) sulfoxide, cyclic non-aromatic ether, straight-chain or branched monoaliphatic alcohol, alkylated formamide, or mixtures thereof. Most preferably, the compatible solute (CS) is selected from polyethylene glycol 400, polyethylene glycol 600, or mixtures thereof, and the organic solvent (OS) is selected from dimethyl sulfoxide, 1,4-di(II,III) ... Alkane, dimethylformamide, methanol, ethanol, or mixtures thereof, The volume ratio of the compatible solute volume Vcs to the organic solvent volume Vos is the same, and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent Vos and the compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos;Vcs)).

[0285] 9. A method for selecting the composition of an immersion solution according to any one of embodiments 1 to 7, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is a polyhydroxy compound or a mixture thereof, and the organic solvent is (alkylated) sulfoxide, cyclic non-aromatic ether, straight-chain or branched monoaliphatic alcohol, alkylated formamide or a mixture thereof. More preferably, the compatible solute (cs) is a polyol or a mixture thereof, and the organic solvent (os) is selected from (alkylated) sulfoxides, cyclic non-aromatic ethers, straight-chain or branched monoaliphatic alcohols, alkylated formamides or mixtures thereof. Most preferably, the compatible solute (cs) is selected from polyols propane-1,2,3-triol (glycerol), ethylene-1,2-diol (ethylene glycol), 2-methylpentane-2,4-diol (2-methylpentane-2,4-diol), (3R,4S,5S,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxacyclohexane-3,4,5-triol (1-glucosylglycerol), or mixtures thereof, and the organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dioxane, and other organic solvents. Alkane, dimethylformamide, methanol, ethanol, or mixtures thereof, The volume ratio of the compatible solute volume Vcs to the organic solvent volume Vos is the same, and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent Vos and the compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos;Vcs)).

[0286] 10. The method for selecting the composition of an immersion solution according to any one of embodiments 1 to 7, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is a polyhydroxy compound a or a mixture thereof, and the organic solvent (os) is a low-reactivity proton or aprotic liquid carbohydrate or a mixture thereof capable of dissolving small molecules. More preferably, the compatible solute (cs) is a monosaccharide, disaccharide, trisaccharide, oligosaccharide, or polysaccharide, or a mixture thereof, and the organic solvent (os) is selected from (alkylated) sulfoxides, cyclic non-aromatic ethers, straight-chain or branched monoaliphatic alcohols, alkylated formamides, or mixtures thereof. Most preferably, the compatible solute cs is selected from sugars (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxacyclohexyl-2-yl]oxyoxacyclohexyl-3,4,5-triol (trehalose) or β-D-fructofuranosyl-α-D-glucopyranoside (sucrose) or mixtures thereof, and the organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dimethyl sulfoxide, etc. Alkane, dimethylformamide, methanol, ethanol, or mixtures thereof, The molar ratio of the compatible solute to the organic solvent is the same, wherein the molar ratio is from 1:10000 to 10000:1, preferably between 100:1 and 1:100, and more preferably between 10:1 and 1:10. The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent Vos and the compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 100:1 and 1:100, and most preferably between 85:15 and 1:1 (Vcrs:(Vos;Vcs)).

[0287] 11. The method for selecting the composition of an immersion solution according to any one of embodiments 1 to 7, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is an L-amino acid or a D-amino acid or a mixture thereof, and the organic solvent (os) is a low-reactivity proton or aprotic liquid carbohydrate or a mixture thereof capable of dissolving small molecules. More preferably, the compatible solute (cs) is an L-amino acid or a mixture thereof, and the organic solvent is (alkylated) sulfoxide, cyclic non-aromatic ether, straight-chain or branched monoaliphatic alcohol, alkylated formamide or a mixture thereof. Most preferably, the compatible solute (cs) is selected from L-amino acid (2S)-pyrrolidine-2-carboxylic acid (proline), 2-aminoacetic acid (glycine), (6S)-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (ectoine), or (5S,6S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (hydroxyectoine), or mixtures thereof, and the organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dimethylaminosulfoxide, and other organic solvents. Alkane, dimethylformamide, methanol, ethanol, or mixtures thereof, The molar ratio of the organic solvent (os) to the compatible solute (cs) is the same (Mos / Mcs), wherein the molar ratio Mos / Mcs is from 1:10000 to 10000:1, preferably from 100:1 to 1:100, and more preferably from 10:1 to 1:10. The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent Vos and the compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 100:1 and 1:100, and most preferably between 85:15 and 1:1 (Vcrs:(Vos;Vcs)).

[0288] 12. The method for selecting the composition of an immersion solution according to any one of embodiments 1 to 7, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (cs) is methylamine or a mixture thereof, and the organic solvent (os) is a low-reactivity proton or aprotic liquid carbohydrate or a mixture thereof capable of dissolving small molecules. More preferably, the compatible solute (cs) is selected from N,N-dimethylamine oxide (trimethylamine N-oxide or TMAO), 2-trimethylammonium acetate (trimethylglycine or betaine), 2-(methylamino)acetic acid (N-methylglycine or sarcosine), or mixtures thereof, and the organic solvent (os) is (alkylated) sulfoxide, cyclic non-aromatic ether, straight-chain or branched monoaliphatic alcohol, alkylated formamide, or mixtures thereof. Most preferably, the compatible solute (cs) is selected from N,N-dimethylamine oxide (trimethylamine N-oxide or TMAO), 2-trimethylammonium acetate (trimethylglycine or betaine), 2-(methylamino)acetic acid (N-methylglycine or sarcosine), or mixtures thereof, and the organic solvent (os) is selected from dimethyl sulfoxide, 1,4-dimethylamine oxide, etc. Alkane, dimethylformamide, methanol, ethanol, or mixtures thereof, The molar ratio of the organic solvent (os) to the compatible solute (cs) is the same (Mos / Mcs), wherein the molar ratio Mos / Mcs is from 1:10000 to 10000:1, preferably from 100:1 to 1:100, and more preferably from 10:1 to 1:10. The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent Vos and the compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 100:1 and 1:100, and most preferably between 85:15 and 1:1 (Vcrs:(Vos;Vcs)).

[0289] 13. The method for selecting the composition of an immersion solution according to any one of embodiments 1 to 8, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereof, an organic solvent (os), and a compatible solute (cs). The compatible solute (CS) is polyethylene glycol 400, and the organic solvent (OS) is dimethyl sulfoxide. The volume ratio of polyethylene glycol 400 (Vcs) to dimethyl sulfoxide (DMS) (Vos) is the same, and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) polyethylene glycol 400 is 1:1000000 to 1000000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos:Vcs)).

[0290] 14. The method for selecting the composition of an immersion solution according to any one of embodiments 1 to 7 and 9, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, as well as an organic solvent (os) and a compatible solute (cs). The compatible solute (cs) is ethylene-1,2-diol (ethylene glycol), and the organic solvent (os) is dimethyl sulfoxide. The volume ratio of ethylene-1,2-diol (ethylene glycol) Vcs to dimethyl sulfoxide (dimethyl sulfoxide) Vos is the same, and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) ethylene-1,2-diol (ethylene glycol) is 1:1000000 to 1000000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos:Vcs)).

[0291] 15. A method for selecting the composition of an immersion solution according to any one of embodiments 1 to 7 and 9, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, as well as an organic solvent (os) and a compatible solute (cs). The compatible solute (cs) is propane-1,2,3-triol (glycerol), and the organic solvent (os) is dimethyl sulfoxide. The volume ratio of propan-1,2,3-triol (glycerol) Vcs to dimethyl sulfoxide (DMSO) Vos is the same, and the volume ratio is 1:1,000,000 to 1,000,000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) propion-1,2,3-triol (glycerol) is 1:1,000,000 to 1,000,000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos:Vcs)).

[0292] 16. The method for selecting the composition of an immersion solution according to any one of embodiments 1 to 7 and 9, wherein the immersion solution comprises a crystallization solution (crs) or an equivalent aqueous solution thereof, as well as an organic solvent (os) and a compatible solute (cs). The compatible solute (cs) is 2-methylpentane-2,4-diol (MPD), and the organic solvent (os) is dimethyl sulfoxide. The volume ratio of 2-methylpentane-2,4-diol (MPD) to dimethyl sulfoxide (DMS) is the same, and the volume ratio is 1:1,000,000 to 1,000,000:1, preferably between 8:1 and 1:8, and more preferably between 3:1 and 1:1 (Vcs:Vos). The ratio of the volume of the crystallization solution or equivalent aqueous solution Vcrs to the combined volume of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) 2-methylpentane-2,4-diol (MPD) is 1:1,000,000 to 1,000,000:1, preferably between 99:1 and 1:99, and most preferably between 85:15 and 1:1 (Vcrs:(Vos:Vcs)).

[0293] 17. The method for selecting the composition of the soaking solution according to any one of embodiments 1 to 16, the method further comprising the following steps:

[0294] 1) Prepare m series of 1n to yn individually soaked solutions by mixing os, cs, crs, and w, wherein m is at least 1, and wherein in each of the m series: a) y is at least 2, b) OS and CRS are the same as in the first series. c) Vos and Vcs are the same as in the first series, or Mos / Mcs are the same as in the first series. d) The cs is changed and is different from the cs of the first series. In each individual solution of each of the m series: e) Vos and Vcs remain unchanged, or Mos / Mcs remains unchanged. f) Vcrs and Vw vary in each solution. In solution 1n, V crs For the minimum V crs (V) crs min), V w For the maximum V w (V) w max). In solution yn, V w For the minimum V w (V) w min), V crs For the maximum V crs (V) crs max). When y > 2, in any additional solution between 1n and yn, V crs In V crs min and V crs The values ​​vary between max and V, and conversely, V w In V w min and V w Variations between max values; 2) Transfer at least one crystal of the biomacromolecule crystal from each compartment to y compartments, each compartment containing one of the solutions prepared in step 1); or transfer each solution prepared in step 1) to y compartments, each compartment containing at least one crystal of the biomacromolecule crystal from its crystallization solution.

[0295] 18. A method for selecting the composition of an immersion solution according to any one of embodiments 1 to 17, wherein an equivalent salt solution different from the crystallization solution is used, wherein the salt is selected from disodium 2,3-dihydroxysuccinate (sodium tartrate), (2R,3R)-2,3-dihydroxysuccinate (dipotassium tartrate), ammonium phosphate (ammonium phosphate), diammonium sulfate (ammonium sulfate), triammonium 2-hydroxypropyl-1,2,3-tricarboxylate (ammonium citrate), sodium acetate (sodium acetate), ammonium acetate (ammonium acetate), dilithium sulfate, lithium chloride, lithium acetate, lithium formate, lithium nitrate, sodium nitrate, sodium chloride, potassium chloride, sodium formate, monophosphates of sodium and potassium, diphosphates and polyphosphates of sodium and potassium, sodium citrate (such as sodium dihydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (monosodium citrate), disodium hydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (monosodium citrate)). The equivalent solution contains sodium, trisodium 2-hydroxypropyl-1,2,3-tricarboxylate (trisodium citrate), magnesium dicarboxylate, magnesium dichloride, magnesium sulfate, calcium dichloride dihydrate, disodium succinate (sodium succinate), cadmium(II) sulfate (cadmium sulfate), disodium malonate (sodium malonate), magnesium diacetate (magnesium acetate), zinc diacetate (zinc acetate), and calcium diacetate (calcium acetate), wherein the salts of the equivalent solution are preferably selected from lithium acetate, lithium chloride, lithium formate, lithium nitrate, dilithium sulfate, sodium chloride, potassium chloride, magnesium dichloride, diammonium sulfate (ammonium sulfate), sodium acetate (sodium acetate), sodium citrate (such as sodium dihydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (monosodium citrate), disodium hydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (disodium citrate), trisodium 2-hydroxypropyl-1,2,3-tricarboxylate (trisodium citrate), magnesium dicarboxylate, and sodium nitrate.

[0296] 19. The method for selecting the composition of the soaking solution according to embodiments 1 to 18, wherein the salt is sodium chloride, potassium chloride, magnesium diacetate (magnesium acetate), and lithium disulfate.

[0297] 20. The method for selecting the composition of the soaking solution according to any one of embodiments 1 to 19, wherein the percentage of the total volume (VT) of each solution in the first series is VT = Vcrs + Vw + Vos + Vcs = 100%.

[0298] 21. The method for selecting the composition of the soaking solution according to any one of embodiments 1 to 20, wherein the step of controlling the crystal is performed by X-ray diffraction, neutron diffraction, absorption or reflection spectra in the ultraviolet and visible spectral regions, infrared spectroscopy and / or visual analysis.

[0299] 22. The method for selecting the composition of an immersion solution according to any one of embodiments 1 to 21, wherein the OS contains small molecules to be analyzed, including small fragments and small probes, capable of diffusing into the biomacromolecule crystals.

[0300] 23. A method for small molecule screening of biological macromolecule crystals, wherein the screening is carried out in an soaking solution selected according to the method described in embodiment 22.

[0301] 24. An immersion solution, said immersion solution being obtained by means of selecting the composition of the immersion solution according to any one of embodiments 1 to 22.

[0302] 25. The soaking solution according to embodiment 24, wherein the Vos is at least 0.5%.

[0303] 26. Use of the soaking solution according to any one of embodiments 24 or 25 for small molecule screening of biomacromolecule crystals.

[0304] 27. An array arranged to perform the method according to any one of embodiments 1 to 23, wherein the array comprises a first dimension of at least two separate soaking solutions (1n to xn) and a second dimension of at least two separate soaking solutions (1m to ym), wherein each of the soaking solutions is located in a separate compartment of the array, wherein each of the soaking solutions comprises an organic solvent (os), a compatible solute (cs), a crystallization solution (crs), and water, wherein in a series of soaking solutions in the first dimension, the ratio of the volume of the compatible solute (Vcs) to the volume of the organic solvent (Vos) is the same, or, in a series of soaking solutions in the first dimension, the ratio of the volume of the organic solvent (os) to the volume of the compatible solute (cs) is the same. The molar ratio Mos / Mcs of the s) are the same, wherein in a series of immersion solutions in the first dimension, the individual immersion solutions in the first dimension contain the same organic solvent and the same compatible solute, wherein in the series of individual immersion solutions in the first dimension, the ratio of water volume Vw to crystallization solution volume Vcrs is varied, wherein one of the individual immersion solutions in the series in the first dimension has a minimum or zero Vcrs and a maximum Vw, and another individual immersion solution in the first dimension has a minimum or zero Vw and a maximum Vcrs, wherein the other individual immersion solutions in the series in the first dimension take Vcrs and Vw values ​​between the two above values.

[0305] 28. The array according to embodiment 27, wherein 1n to xn defines the range from 1 (1n) to 1 × 10 6 The number of individual soaking solutions within the range of (xn), wherein the number includes, but is not limited to, 1×10 6 Or 1×10 5 1×10 4 The array configuration may be 768, 384, 96, 48, or 24; however, the array configuration determines the number of individual soaking solutions.

[0306] 29. The array according to embodiments 27 and 28, wherein the array comprises a second dimension (dII) of a series of individual soaking solutions of biomacromolecule crystals, wherein the compatibility solute varies in the second dimension and depends on the compatibility solute used in the series of soaking solutions in the second dimension, wherein the ratio of the compatibility solute volume Vcs to the organic solvent volume Vos or the molar ratio of the organic solvent (os) to the compatibility solute (cs) Mos / Mcs may be different.

[0307] 30. An array according to any one of embodiments 27 to 29, wherein in each of the series of individual soaking solutions, the ratio of the volume of the compatible solute Vcs to the volume of the organic solvent Vos is the same, the volume ratio being 1:1,000,000 to 1,000,000:1, preferably between 8:1 and 1:8, more preferably between 3:1 and 1:1 (Vcs:Vos).

[0308] 31. An array according to any one of embodiments 27 to 30, wherein in each of the series of individual soaking solutions, the molar ratio of organic solvent (os) to compatible solute (cs) is the same (Mos / Mcs), said molar ratio (Mos / Mcs) being 1:10000 to 10000:1, preferably between 100:1 and 1:100, more preferably between 10:1 and 1:10.

[0309] 32. Use of an array according to any one of embodiments 27 to 31 for obtaining a solution according to any one of embodiments 22 or 23. Attached Figure Description

[0310] Figure 1: 1A is a photograph of the screening plate used for immersion selection; Figure 1B Details of one hole in a 24-well plate are shown. Figure 1C The diagram above illustrates the pores during the immersion experiment.

[0311] Figure 2 A method for selecting an immersion solution according to the present invention is shown, the immersion solution comprising: CS, a compatible solute; OS, an organic solvent; CB (crs), a crystallization buffer (solution); and W, water. Rows (solute A to solute D) represent the first dimension, and columns (1 to 6) represent the second dimension. Alternatively, rows (solute A to solute D) may represent the second dimension, and columns (1 to 6) may represent the first dimension.

[0312] Figure 3An example of a scheme for selecting the soaking solution according to the method of the present invention is shown, including fragment screening of PKA. PEG400, polyethylene glycol 400; MPD, 2-methylpentane-2,4-diol; EG, ethylene-1,2-diol (ethylene glycol); Gly, propylene-1,2,3-triol (glycerol); OS, organic solvent = DMSO; CB (crs), crystallization buffer (solution); W, water.

[0313] Figure 4 The crystal in Figure 3 Photographs after 24 hours of soaking in the solution

[0314] Figure 5 This illustrates an example of the visual difference between suitable crystals (A) and crystals (B) that have suffered severe mass loss due to unfavorable immersion conditions / solution.

[0315] Figure 6 A template for visually examining a crystal under a microscope is shown.

[0316] Figure 7 This section illustrates standards for visual inspection and for controlling and selecting solutions that have the least impact on crystals.

[0317] Figure 8 This shows the PKA crystal in... Figure 3 Visual inspection results after 24 hours of immersion in the test solution. Each series was prepared with different compound solutes: PEG400; MPD; EG, ethylene-1,2-diol (ethylene glycol) or (glycerol); S&C represents surface and cracks; EDG represents edges; COL represents color; SMS represents smooth surface; SME represents smooth edges; Y represents present but few; Y+ represents many; LCR represents longitudinal cracks; CCC represents crisscrossing cracks; ROS represents rough surface; FRE represents worn edges; N represents none; DIS represents dissolution; X represents no data.

[0318] Figure 9 An image of the diffraction pattern after an X-ray experiment is shown.

[0319] Figure 10 A template for X-ray examination of a crystal is shown.

[0320] Figure 11 This shows the standards used for X-ray examination and for controlling and selecting solutions that have the least impact on crystals.

[0321] Figure 12 The crystal in Figure 3 The X-ray examination results after 24 hours of immersion in the solution. RES represents resolution; MOS represents mosaicism; TWI represents twinning; ICE represents ice rings.

[0322] Figure 13 The most suitable soaking conditions for the described scheme are highlighted, thus providing a range of appropriate soaking parameters. An asterisk indicates one of the soaking conditions selected for subsequent fragment selection.

[0323] Figure 14 A template scheme for fragment filtering is shown.

[0324] Figure 15 It shows the use of Figure 13 One of the highlighted components (PEG 400, column 3) is a possible scheme for fragment screening of PKA.

[0325] Figure 16 It shows the use of Figure 13 One of the highlighted components (PEG 400, column 1) (marked with an asterisk) represents a possible scheme for PKA fragment screening. "Frag" followed by a number at the end indicates a single fragment.

[0326] Figure 17 An example of a possible workflow is shown, illustrating the different steps of the method of the present invention performed in the immersion selection experiment and subsequent fragment screening.

[0327] Figure 18 It shows Figure 16 The hit distribution on the example board is shown. The board comprises the first 24 of 87 fragments. Five fragments on this board were proven to be PKA conjugates (see boxes A1, A4, B6, D3, and D5). RES and MOS represent the quality of the dataset in terms of resolution (RES) and mosaicism (MOS).

[0328] Figure 19 A 3D model of PKA is shown, representing the protein and a superposition of all identified binders distributed at different locations on the protein. Enlarged views within circles show the ATP pocket binder clusters in more detail.

[0329] Figure 20 An excerpt of the reconstructed electron density is shown.

[0330] Figure 21 As shown Figure 20 The process is the same, but it utilizes water molecules (spheres) with fixed protein amino acid sequences and structures.

[0331] Figure 22 As shown Figure 20 The same, but utilizing the unallocated electron density of the highlighted (elliptical) representation of small molecule fragment hits.

[0332] Figure 23 As shown Figure 20 Same, but using representation Figure 19 This is accomplished by highlighting the corresponding small molecule fragments in the previously unallocated electron density.

[0333] Figure 24 The excerpt of the structure is shown, illustrating the binding mode of small molecule fragments with the amino acid side chains of the protein and the structurally fixed water (dots) hydrogen bonds (dashed lines; numbers indicate hydrogen bond lengths). Detailed Implementation

[0334] Example

[0335] Example 1: Screening of 87 fragments using protein kinase A (PKA) crystals

[0336] - PKA crystals

[0337] The PKA solution was concentrated to 8–10 mg / mL by filtration and centrifugation. Simultaneously, a buffer exchange was performed with 100 mM Mes-Bis-Tris buffer (pH 6.9) containing 1 mM DTT, 0.1 mM EDTA (ethylenediaminetetraacetic acid), and 75 mM LiCl. The solution was then sterilely filtered. 72 μL of this solution was mixed with 8 μL of 1 M Mes-Bis-Tris buffer (pH 6.9) and 2 μL of 10 mM Mega 8 solution and centrifuged for 15 min. Crystallization was then performed at 4 °C using a 3 μL master mixture as a bottom drop against a 400 μL methanol / water solution with a methanol concentration of 14–23% (v / v).

[0338] - Selection of a suitable soaking solution

[0339] Crystals are collected via a low-temperature ring and transferred to experimental plate wells prepared according to the method of the present invention and distributed in two dimensions. Figure 3 ).

[0340] The first series of six separate soaking solutions were prepared by mixing OS, W, CRS, and CS. DMSO at a concentration of 10% (Vos) was used as the organic solvent. Compatible solutes were 25% polyethylene glycol 400 (PEG 400) and 2-methylpentane-2,4-diol (MPD) and 10% ethylene-1,2-diol (ethylene glycol; EG) or propylene-1,2,3-triol (glycerol; Gly). According to the method of the invention, a crystallization buffer (…) was used… Figure 3 The third component is a diluted solution of CB (crs) in water (W).

[0341] The series in row A prepared using PEG400 as cs can be designated as the first series according to the invention. Each soaking solution in the first series contains the same organic solvent (os) (DMSO) and the same compatible solute (cs), with Vos 10% (1 μl) and Vcs 25% (2.5 μl) remaining constant in all solutions of the series. However, the volume / ratio Vcrs (CB) and Vw vary in each solution of the first series, with one solution consisting of Vwmin 0% (0 μl) and Vcrsmax 65% (6.5 μl) (see column 1), another consisting of Vcrsmin 0% (0 μl) and Vwmax 65% (6.5 μl) (see column 6), and in other solutions (see columns 2-5), Vw varies between Vwmin and Vwmax, and conversely, Vcrs varies between Vcrsmin and Vcrsmax. In this embodiment, in any additional solution, Vcrs gradually varies between Vcrsmin and Vcrsmax, and conversely, Vw gradually varies between Vwmin and Vwmax (see columns 2-5 of the PEG400 row). In any additional solution, the increment of Vw between Vwmin and Vwmax and the increment of Vcrs between Vcrsmin and Vcrsmax remain the same, i.e., 13% (1.3 μl), across each solution in the first series.

[0342] The solution is distributed in one row of the array ( Figure 3 The solution is located in the holes of row A, thus forming the first dimension. The solution is preferably distributed in order from the lowest Vw to the highest Vw, but the distribution order in the array can be random.

[0343] Then, or simultaneously, perform the following steps:

[0344] An additional series of six separate soaking solutions were prepared by mixing OS, W, CRS (CB), and CS. Figure 3In this embodiment, the number of individual soaking solutions, 6, remains constant and is the same as the number of individual soaking solutions in the first series. In the additional series (row B), os is the same as in the first series (DMSO), but cs is changed (row B: BMPD). Within the additional MPD series, each soaking solution in the first series contains the same organic solvent (os) (DMSO) and the same compatible solute (cs) (MPD), and Vos and Vcs remain the same in all solutions of the series. However, the volume / ratio Vcrs (CB) and Vw vary in each solution of the series. One solution consists of Vwmin at 0% (0 μl) and Vcrsmax at 65% (6.5 μl) (see column 1), another consists of Vcrsmin at 0% (0 μl) and Vwmax at 65% (6.5 μl) (see column 6), and in the other solutions (see columns 2-5 of row B), Vw varies between Vwmin and Vwmax, and conversely, Vcrs varies between Vcrsmin and Vcrsmax. In this embodiment, Vcrs varies gradually between Vcrsmin and Vcrsmax, and conversely, Vw varies gradually between Vwmin and Vwmax (see columns 2-5 of row B). The increment of Vw between Vwmin and Vwmax and the increment of Vcrs between Vcrsmin and Vcrsmax remain the same between each solution, i.e., 13% (1.3 μl). In this embodiment, the number of individual soaking solutions in the MPD series (row B), i.e., 6, is the same as the number x of individual soaking solutions in the first series (row A); in the first (PEG400) series and the MPD series, the increment of Vcrs between Vcrsmin and Vcrsmax and the increment of Vw between Vwmin and Vwmax are the same.

[0345] The solution is distributed in an additional row of the array ( Figure 3 The solution is located in the holes of row B, thus forming the second dimension. The solution is preferably distributed in order from the lowest Vw to the highest Vw, but the distribution order in the array can be random.

[0346] exist Figure 3On the same array (but another array may be used), another “first” series (the C-th row using EG, series C) and its additional series (the D-th row using Gly, series D) are prepared and distributed. The series in the C-th row prepared with EG as cs can be designated as the first series according to the invention. Each soaking solution in series C contains the same organic solvent (os) (DMSO) and the same compatible solute (EG), and in all solutions of series C, Vos 10% (10 μl) and Vcs 10% (10 μl) remain the same. However, the volume / ratio Vcrs (CB) and Vw vary in each solution of the first series. One solution consists of Vwmin of 0% (0 μl) and Vcrsmax of 80% (8 μl) (see column 1), another solution consists of Vcrsmin of 0% (0 μl) and Vwmax of 80% (8 μl) (see column 6), and in the other solutions (see columns 2-5), Vw varies between Vwmin and Vwmax, and conversely, Vcrs varies between Vcrsmin and Vcrsmax. In this embodiment, in any additional solution, Vcrs gradually varies between Vcrsmin and Vcrsmax, and conversely, Vw gradually varies between Vwmin and Vwmax (see columns 2-5 in row C). In any additional solution, the increment of Vw between Vwmin and Vwmax and the increment of Vcrs between Vcrsmin and Vcrsmax remain the same, i.e., 16% (1.6 μl), across each solution in the first series.

[0347] The solution is distributed in one row of the array ( Figure 3 The solution is located in the holes of row C, thus forming the first dimension. The solution is preferably distributed in order from the lowest Vw to the highest Vw, but the distribution order in the array can be random.

[0348] Simultaneously or at different times, the following steps are performed.

[0349] An additional series of six separate soaking solutions were prepared by mixing OS, W, CRS (CB: crystallization buffer), and CS. Figure 3In this embodiment, the number of individual soaking solutions, 6, remains constant and is the same as the number of individual soaking solutions in the first series. In the additional series (row D, series D), os is the same as in the first series (DMSO), but cs is changed (Gly). Within series D, each soaking solution in the first series contains the same organic solvent (os) (DMSO) and the same compatible solute (cs) (Gly), and Vos and Vcs remain the same in all solutions of series D. However, the volume / ratio Vcrs (CB) and Vw vary in each solution of the D series. One solution consists of Vwmin at 0% (0 μl) and Vcrsmax at 80% (8 μl) (see column 1), another solution consists of Vcrsmin at 0% (0 μl) and Vwmax at 80% (8 μl) (see column 6), and in the other solutions (see columns 2-5 of row D), Vw varies between Vwmin and Vwmax, and conversely, Vcrs varies between Vcrsmin and Vcrsmax. In this embodiment, Vcrs varies gradually between Vcrsmin and Vcrsmax, and conversely, Vw varies gradually between Vwmin and Vwmax (see columns 2-5 of row D). The increment of Vw between Vwmin and Vwmax and the increment of Vcrs between Vcrsmin and Vcrsmax remain the same in each solution, i.e., 16% (1.6 μl). In this embodiment, the number of individual soaking solutions in the MPD series (row D), i.e., 6, is the same as the number x of individual soaking solutions in the first series (row C); in the first (EG) series and the Gly series, the increment of Vcrs between Vcrsmin and Vcrsmax and the increment of Vw between Vwmin and Vwmax are the same.

[0350] Then, place at least one PKA crystal prepared in a) above into Figure 3 In each compartment of the array. The crystals are placed under a microscope for visual analysis and control.

[0351] After transferring the crystal into each well, each well was photographed for visual control, and the existing crystals were characterized immediately after transfer (0 hours), 1 hour, and 24 hours later. A photograph of the crystal after 24 hours is shown below. Figure 4 As shown, the visual inspection results of the surface, edges, and color are presented in... Figure 8 (Data for 0 hours and 1 hour later are not shown). Figure 4It is clearly shown that after 24 hours, crystals were still present in wells 1-6 of row A, wells 1-4 of row B, and well 3 of row C. Some crystal residues were visible in wells 5 and 6 of row B and wells 2 and 4 of row C, showing signs of pre-dissolution. No crystals were left in wells 1, 5, and 6 of row C and in row D.

[0352] Based on visual inspection of the crystals, a suitable solution for soaking the crystals was selected. The crystals in wells 1-4 of row A (PEG400) and wells 1 and 2 of row B (MPD) appeared suitable for diffraction testing. Figure 4 and Figure 8 Based on visual inspection, the crystals in wells 5 and 6 of row A, wells 3 and 4 of row B, and the crystals in row C were not expected to diffract, therefore these solutions were not selected.

[0353] For further control and selection, crystals were collected using a cryogenic ring, vitrified in liquid nitrogen, and subjected to internal X-ray measurements. For each crystal, two perpendicular diffraction patterns were measured (at positions 0° and 90°). Figure 12 The results shown demonstrate good diffraction quality of the crystals obtained from wells 1 through 4 in row A and well 2 in row B. As expected, the crystals from wells 5 and 6 in row A, wells 3 and 4 in row B, and the crystals in row C did not show diffraction. Unexpectedly, the crystal from well 1 in row B also did not show diffraction. The selection of the compatibility solute, organic solvent, and concentration range proved suitable for determining the range of parameters affecting the integrity, stability, and quality of protein crystals. Figure 13 The appropriate conditions obtained according to the scheme have been plotted and highlighted. The plot shows multiple appropriate conditions (4 solutions) when using PEG400 and one condition when using MPD.

[0354] - Filtering PKA segments.

[0355] Regarding the screening of PKA fragments, the composition of the soaking solution in the first well of row A was selected (in... Figure 13 (Indicated by asterisks). Immersion solutions with equal proportions of CS (25% PEG400), CRS (65% CB), and OS (10% DMSO) were prepared. Several 24-well drop plates were filled with the solutions. Figure 16 DMSO containing 1 M fragments (final concentration of 100 mM in each well) was used instead of DMSO alone. The newly prepared crystals were then transferred into the wells using a low-temperature ring. In total, the experiment subjected the PKA crystals to 87 different fragments.

[0356] exist Figure 15Above, another example of a screening plate is shown, using a selected composition of a suitable alternative soaking solution consisting of 25% CS (PEG400), 39% CS (CB), 26% water, and 10% DMSO, selected according to the method of the invention. See [link to image]. Figure 13 The third column of row A.

[0357] After a 24-hour immersion period, the immersed crystals were collected using a cryogenic ring and vitrified in liquid nitrogen. Data collection was performed on a synchrotron. At a resolution of 1.3–1.8 Å, data evaluation yielded 55 conjugates. Figure 18 It shows Figure 16 The hit distribution on the example board. Refinement of the corresponding data yielded a 3D model of 55 fragments interacting with PKA at different interaction sites, of which 27 fragments bound to the ATP-binding pocket. Figure 19 The superposition of proteins and their corresponding conjugates is shown. This study is merely one example of the highly successful application of the method of the present invention to determine the soaking parameters for small molecule screening (fragments in this example). Several unexpected chemical types of kinase conjugates were hit. This demonstrates that the method of the present invention is a highly successful means of performing rapid and reliable small molecule screening and providing high-quality data.

[0358] Example 2: A rule-based method for selecting suitable soaking solutions and subsequent small molecule screening

[0359] In order to perform small fragment screening, it is necessary to obtain crystals of the target protein (POI). Figure 17 The workflow involves step a). Crystallization is carried out using a commercially available 24-well drop crystallization plate (or any other plate form) according to a vapor diffusion method (Figure 1). For this purpose, the reservoir of each 24-well plate is filled with buffer according to a predetermined crystallization protocol. Furthermore, crystallization droplets are prepared in the grooves at the top of each well in a column by adding a volume of protein solution to a volume of the same buffer used for the reservoir. Typically, but not always, the volume ratio is 1:1 (Figure 1). The resulting protein concentration is typically, but not always, in the range of 2 to 30 mg / ml, but can also reach concentrations above 250 mg / ml. The wells are then sealed with an adhesive membrane called sealing tape. All these steps are performed according to the crystallization protocol in a properly conditioned laboratory, typically at 4°C, 10°C, 18°C, or 20°C. The time required to store the plate for crystallization on a shelf in the laboratory is typically several days to several weeks.

[0360] After the crystallization process is completed, the soaking solution is selected according to the present invention to plot the conditions for screening small molecule fragments (at the same temperature). For this purpose, a 24-well crystallization plate (or any other plate form) is used, and the well of each well is provided with a possible soaking solution prepared according to the present invention, as shown in Figures 1 to 14. Figure 3 As shown ( Figure 17 Step b) of the workflow. Subsequently, the membrane used to seal the 24-well plate is removed from the plate used for POI crystallization. Using specialized laboratory tools typically equipped with crystal-picking nylon rings or any other suitable so-called "crystal catching" tools, the simultaneously grown crystals are removed from their growth solution and transferred to a possible soaking solution in the grooves of a new 24-well plate prepared according to the example of the described scheme. Figure 17 Step c in the workflow. These plates are also sealed with sealing tape.

[0361] The next step aims to control crystal quality, maximum soaking time, and the appropriate relationship between soaking time and crystal quality for small molecule fragment screening. Within a certain timeframe, typically the next 24 hours, photographs of each groove are taken under a microscope at least once, usually three times, and sometimes more, to record crystal conditions under the microscope, such as immediately after transfer, 1 hour later, and 24 hours later. Figures 4 to 5 At the same time, annotations are usually added to supplement the information in the photos. Figure 6 Examples illustrate templates for this type of annotation. Annotations refer to the different types of cracks a crystal may exhibit (longitudinal, transverse, crisscrossing), the appearance of the crystal surface (e.g., smooth, rough), or the appearance of its edges (e.g., sharp, rounded) and their properties under polarized light (strong color, colored, colorless). Figure 8 (Taking 24 hours as an example). In most cases, not all crystals can withstand all conditions. Some crystals will dissolve or break down over time, while others will remain in good condition. Therefore, X-ray evaluation of crystal quality is recommended. Figure 17 Step d) of the workflow. For this purpose, the sealing tape is removed from the plate to allow the remaining crystals on the plate to be transferred to liquid nitrogen or another vitrification medium. In the case of X-ray studies at room temperature, prior vitrification is not required. Vitrification of the crystals occurs in the aforementioned nylon ring or any other equivalent transfer device such as a rock ring or capillary. Crystals vitrified in a nylon ring or other transfer device are stored in so-called “cryogenic vials” in liquid nitrogen or another vitrification medium until they are subjected to X-ray examination. Crystals can also be stored in any other suitable storage container.

[0362] For X-ray examination, the crystals in a nylon ring or other transfer device are removed from the cryogenic vial or other storage container and fixed in a so-called cryogenic stream on the X-ray machine to maintain the temperature around the protein crystals. In the case of room temperature X-ray studies, a cryogenic stream is not required. X-ray data is then collected. Using an internal X-ray source, two to several images are collected. Due to its rapid data acquisition, the entire dataset is collected on a synchrotron source (preferably per...). Figures 10 to 11Templates). Regarding resolution and mosaicism, the appearance of artifact data originating from unwanted ice crystals in protein crystals, and the phenomenon sometimes referred to as "twin crystals" ( Figure 5 B) Regarding the original data ( Figure 12 The evaluation is conducted. With data collected at the synchrotron, experienced crystallographers determine whether the appearance of the so-called "electron density" is appropriate. Based on this data, a decision is made regarding which immersion conditions(s) to select for small molecule fragment screening. Figure 13 Mark with an asterisk; Figure 17 (e / g) of the workflow. In contrast to traditional soaking solution optimization that yields a single soaking condition, the rule-based method of this invention typically provides a range of soaking conditions. Experimenters can select from this series of alternative soaking solutions to address potential adverse effects on crystals caused by, for example, the specific properties of small molecular fragments or pH variations.

[0363] Optionally, stress testing can be performed at this stage of the workflow. Figure 17 Step f in the workflow. For this purpose, one or more previously obtained soaking solutions are selected and prepared in new 24-well plates. The crystals are then transferred to these solutions. For stress testing, small molecules dissolved in DMSO, such as molecular probes or small molecule fragments, are used instead of pure DMSO. They apply different kinds of stress to the crystals, such as pH stress or osmotic stress. Estimates are then made based on subsequent data (and...). Figure 6 (The process steps are the same as step d in the workflow), and one or more final soaking solutions can be selected.

[0364] Typically, a soaking condition is determined for subsequent screening of small molecule fragments. For screening of 300 small molecule fragments, 13 wells of a 24-well plate are prepared by pipetting the corresponding soaking solution composition of the selected DMSO-free soaking solution. Instead of pure DMSO, 300 small molecules are typically dissolved in DMSO at a concentration of 1 M and added to the prepared 300 wells. This results in a final concentration of the small molecule fragment in each soaking solution that is typically 100 mM, but may be higher or lower. Figures 15 to 16 ; Figure 17 Step h in the workflow. Afterwards, typically two newly prepared crystals (sometimes only one or more) are transferred to each soaking solution and soaked for approximately 24 hours. Figure 17 Step i) of the workflow. Then, the crystal is captured and transferred to liquid nitrogen for vitrification ( Figure 17 Step j of the workflow), stored in cryogenic vials in Uni-pucks or some other suitable storage device, and sent to the synchrotron facility for data collection. Figure 17Step k of the workflow. In the case of room temperature X-ray studies, vitrification is not required.

[0365] During data collection, separate datasets for all crystals were obtained, including hundreds to thousands of photographs showing X-ray beam reflections (such as...). Figure 9 (As shown). These reflections originate from the interaction between X-rays and electrons, and can therefore be used to reconstruct the so-called electron density. This electron density defines the space in which electrons reside within a crystal and represents the geometry of a protein. Computational methods are used to extract important information from these datasets. To reconstruct the geometry, other information, the so-called phases, is needed, which cannot be extracted from such experiments without heavy atom derivatization. This information can be obtained from the structural resolution of previously obtained identical or very similar proteins using a method called molecular substitution. The result is a 3D model of the protein geometry corresponding to the positions of all electrons in the protein ( Figure 19 The electron density was combined with the known amino acid sequence of the corresponding protein, and the location of the structurally fixed water molecule was identified. This structural resolution must be optimized over several cycles of structural refinement to obtain a convincing fit between the measurement data and the model. The result is an atomic analytical representation of the target geometry. Figure 19 ; Figure 17 Step 1 of the workflow.

[0366] Each of these structures was examined to identify specific small molecule fragments that could be assigned to the crystals used for soaking in this particular crystal. Figures 21 to 22 electron density () Figure 20 If this is the case, the small molecule fragment is considered a "hit." An examination of all datasets revealed all small molecule fragment conjugates ( Figure 23 The hit count of the experimental layout on a screening plate was plotted, covering five hits. Datasets without additional electron density that could be assigned to the corresponding small molecule fragments were discarded. Further refinement of each of these "hit" datasets revealed the precise geometry of the interactions between the protein and the corresponding small molecule fragment. Figure 24 Therefore, the process provides validated knowledge about small molecule fragments binding to protein targets and elucidates structurally defined binding modes that can be used in medicinal chemistry optimization processes. Larger drug-sized molecules can also be used, for example, to validate subsequent compounds derived from medicinal chemistry optimization. Furthermore, specialized molecular probes can be used to characterize the nature of binding sites or obtain information about putative pharmacophore patterns, etc.

Claims

1. A method for selecting an immersion solution for immersing biomacromolecules in crystalline form, wherein the immersion solution comprises an organic solvent, a compatible solute, a crystallization solution, and / or water; the method comprising the following steps: 1) A first series of 1n to xn individual soaking solutions are prepared by mixing the organic solvent, compatible solute, crystallization solution, and water, wherein: a) x is at least 2, b) Each soaking solution in the first series contains the same organic solvent, the same compatible solute, and the same crystallization solution. c) The volume of organic solvent and the volume of compatible solute are the same in every solution, or the molar ratio of organic solvent to compatible solute is the same in every solution. d) The volume of the crystallizing solution and the volume of water vary in each solution. In solution 1n, the volume of the crystallizing solution is the minimum volume of the crystallizing solution, and the volume of water is the maximum volume of water. In solution xn, the volume of water is the minimum volume of water, and the volume of the crystallizing solution is the maximum volume of the crystallizing solution. When x>2, in any additional solution between 1n and xn, the volume of the crystallization solution varies between the minimum and maximum crystallization solution volume, and conversely, the volume of water varies between the minimum and maximum water volume; 2) Transfer at least one crystal of the biomacromolecule from each compartment to x compartments, each compartment containing one of the solutions prepared in step 1); or transfer each solution prepared in step 1) to x compartments, each compartment containing at least one crystal of the biomacromolecule from its crystallization solution. 3) Control the crystals in each compartment; 4) Select a suitable soaking solution for soaking the crystals; The compatible solute is selected from polyhydroxy compounds, amino acids, methylamine, and mixtures thereof. The organic solvent is selected from low-reactivity proton or aprotic liquid carbohydrates and mixtures thereof capable of dissolving small molecules, the crystallizing solution is obtained from the crystallization process of the crystal, or is equivalent to it in terms of net osmotic pressure, dielectric constant, solubility of the corresponding biomacromolecule, and effect on the structural conformation of the corresponding biomacromolecule, and contains pH and buffering substances, additives and precipitants.

2. The method according to claim 1, wherein the compatible solute is a polyhydroxy compound or a mixture thereof.

3. The method according to claim 1, wherein the organic solvent is an alkyl sulfoxide, a cyclic non-aromatic ether, a straight-chain or branched monoaliphatic alcohol, an alkyl formamide, or a mixture thereof.

4. The method according to claim 1, wherein the compatible solute is a monosaccharide, oligosaccharide, or polysaccharide or a mixture thereof.

5. The method according to claim 1, wherein the compatible solute is a disaccharide or a trisaccharide.

6. The method according to claim 1, wherein the compatible solute is at least one methylamine.

7. The method according to claim 1, wherein the compatible solute is polyethylene glycol 400.

8. The method according to claim 1, wherein the compatible solute is ethylene-1,2-diol (ethylene glycol).

9. The method according to claim 1, wherein the compatible solute is propan-1,2,3-triol (glycerol).

10. The method according to claim 1, wherein the compatible solute is 2-methylpentane-2,4-diol (MPD).

11. The method according to any one of claims 1 to 10, the method further comprising the following steps: 1) Prepare m series of 1n to yn individually soaked solutions by mixing the organic solvent, compatible solute, crystallization solution, and water, wherein m is at least 1, and wherein in each of the m series: a) y is at least 2, b) The organic solvent and crystallization solution are the same as those in the first series. c) The volume of organic solvent and the volume of compatible solute are the same as in the first series, or the molar ratio of organic solvent to compatible solute is the same as in the first series. d) The compatibility of the solute changes and is different from that of the first series of compatible solutes. In each individual solution of each of the m series: e) The volumes of the organic solvent and the compatible solute remain constant, or the molar ratio of the organic solvent to the compatible solute remains constant. f) The volume of the crystallization solution and the volume of water vary in each solution. In solution 1n, the volume of the crystallizing solution is the minimum volume of the crystallizing solution, and the volume of water is the maximum volume of water. In solution yn, the volume of water is the minimum volume, and the volume of the crystallizing solution is the maximum volume. When y>2, in any additional solution between 1n and yn, the volume of the crystallization solution varies between the minimum and maximum crystallization solution volumes, and conversely, the volume of water varies between the minimum and maximum water volumes. 2) Transfer at least one crystal of the biomacromolecule from each compartment to y compartments, each compartment containing one of the solutions prepared in step 1); or transfer each solution prepared in step 1) to y compartments, each compartment containing at least one crystal of the biomacromolecule from its crystallization solution.

12. The method according to any one of claims 1 to 10, wherein an equivalent salt solution different from the crystallization solution is used, wherein the salt is selected from disodium 2,3-dihydroxysuccinate (sodium tartrate), (2R,3R)-2,3-dihydroxysuccinate (dipotassium tartrate), ammonium phosphate (ammonium phosphate), diammonium sulfate (ammonium sulfate), triammonium 2-hydroxypropyl-1,2,3-tricarboxylate (ammonium citrate), sodium acetate (sodium acetate), ammonium acetate (ammonium acetate), dilithium sulfate, lithium chloride, lithium acetate, lithium formate, lithium nitrate, sodium nitrate, sodium chloride, potassium chloride, sodium formate, monophosphates of sodium and potassium, diphosphates and polyphosphates of sodium and potassium, sodium citrate, magnesium diformate, magnesium dichloride, magnesium sulfate, calcium dichloride dihydrate, disodium succinate (sodium succinate), CdSO4, disodium malonate (sodium malonate), magnesium diacetate (magnesium acetate), zinc diacetate (zinc acetate), calcium diacetate (calcium acetate).

13. The method according to claim 12, wherein the sodium citrate comprises sodium dihydrogen 2-hydroxypropyl-1,2,3-tricarboxylate, disodium 2-hydroxypropyl-1,2,3-tricarboxylate, and trisodium 2-hydroxypropyl-1,2,3-tricarboxylate.

14. The method according to any one of claims 1 to 10, wherein a solution equivalent to the crystallization solution is used, and the salt of the solution equivalent to the crystallization solution is selected from lithium acetate, lithium chloride, lithium formate, lithium nitrate, dilithium sulfate, sodium chloride, potassium chloride, magnesium dichloride, diammonium sulfate, sodium acetate, sodium dihydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (monosodium citrate), disodium hydrogen 2-hydroxypropyl-1,2,3-tricarboxylate (disodium citrate), trisodium 2-hydroxypropyl-1,2,3-tricarboxylate (trisodium citrate), magnesium dicarboxylate, and sodium nitrate.

15. The method according to any one of claims 1 to 10, wherein the organic solvent contains small molecules to be analyzed, including small fragments and small probes, diffused into the biomacromolecule crystal.

16. The method of claim 1, wherein x is 2 to 1 × 10⁻⁶. 6 .

17. The method of claim 11, wherein y is 2 to 1 × 10⁻⁶. 6 .

18. The method according to claim 1, wherein the organic solvent is dimethyl sulfoxide.

19. The method according to claim 1, wherein the compatible solute is polyethylene oxide.

20. The method of claim 1, wherein the volume ratio of the compatible solute to the organic solvent is the same, and the volume ratio is from 1:1000000 to 1000000:

1.

21. The method according to claim 1, wherein the ratio of the volume of the crystallization solution or equivalent salt solution to the combined volume of the organic solvent and the compatible solute is from 1:1000000 to 1000000:

1.

22. The method according to claim 1, wherein the organic solvent is 1,4-di(II) ... alkyl.

23. The method according to claim 1, wherein the compatible solute is one or more amino acids.

24. The method according to claim 1, wherein the compatible solute is one or more amino acids selected from the group consisting of: 2-aminoethanesulfonic acid (taurine), 2-aminoethanesulfinic acid (hypotaurine), (2S)-pyrrolidine-2-carboxylic acid (proline), 2-aminoacetic acid (glycine), (6S)-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (ectoine), (5S,6S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-6-carboxylic acid (hydroxyectoine), and 4-aminobutyric acid (γ-aminobutyric acid). (2S)-2-aminoglutaric acid (glutamic acid), β-hydroxy-γ-N-trimethylaminobutyric acid, (2S)-2-amino-3-methylbutyric acid (valine), (2S,3S)-2-amino-3-methylvaleric acid (isoleucine), (2S)-2-aminosuccinic acid (aspartic acid), (2S)-2-aminopropionic acid (alanine), 2-(methylamino)ethanesulfonic acid (N-methyltaurine), (2S)-2-[[(1R)-1-carboxyethyl]amino]-5-(diaminomethyleneamino)valeric acid (Octopin) and mixtures thereof.

25. The method according to claim 1, wherein the compatible solute is a polyol.

26. The method according to claim 1, wherein the compatible solute is one or more sugars selected from the group consisting of (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxecyclohexyl-2-yl]oxyoxecyclohexyl-3,4,5-triol (trehalose), (2S,3R,4R,5R)-hexyl-1,2,3,4,5,6-hexane (sorbitol), and β-D-fructofuranosyl-α-D-glucopyranoside (sucrose).

27. The method according to claim 1, wherein the compatible solute is one or more methylamines selected from the following: N,N-dimethylmethylamine oxide (trimethylamine N-oxide or TMAO), 2-trimethylammonium acetate (trimethylglycine or betaine), 2-(methylamino)acetic acid (N-methylglycine or sarcosine), 2-hydroxyethyl(trimethyl)ammonium (choline), 2-[formamidinyl(methyl)amino]acetic acid (creatine), L-α-glycerophosphorylcholine, (R)-(3-carboxy-2-hydroxypropyl)-N,N,N-trimethylammonium hydroxide (carnitine).

28. The method of claim 1, wherein the organic solvent is selected from linear or branched monoaliphatic alcohols; dialiphatic or polyaliphatic alcohols; monoalkyl ethers of aliphatic diols; dialkyl ketones; cyclic nonaromatic ethers; cycloalkenyl ethers; benzyl alcohol; phenethyl alcohol; alkylated sulfoxides; esters derived from acetic acid; alkylated formamides; organic nitriles; lactones; polyethers; polyetheramines; and sugar ethers.

29. The method according to claim 1, wherein the organic solvent is selected from methanol, ethanol, propan-1-ol, propan-2-ol, 2-methylpropan-1-ol (isobutanol), butan-1-ol (n-butanol), 2-methylpropan-2-ol (tert-butanol), 2-methyl-2,4-pentanediol, hexane-2,5-diol (2,5-hexanediol), propan-1,3-diol (1,3-propanediol), butan-2,3-diol, propan-1,2-diol (1,2-propanediol), ethylene-1,2-diol (ethylene glycol), monomethyl ether of ethylene glycol, propan-2-one (acetone), oxapentane (tetrahydrofuran), crown ether, 1,4-di... Alkane (di) Alkane), furan, methyl sulfoxide (DMSO), ethyl acetate, N,N-dimethylformamide, acetonitrile, oxepane-2-one (γ-butyrolactone), 4-methyloxepane-2-one (β-butyrolactone), polyethylene glycol, Jeffamine, 2-(2-ethoxyethoxy)ethanol (diethylene glycol).

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