SAMPLE HOLDER AND SAMPLE PREPARATION METHOD USING SAID SAMPLE HOLDER

Description

SAMPLE HOLDER AND SAMPLE PREPARATION METHOD USING SAID SAMPLE HOLDER DESCRIPTION FIELD

[0001] The present description is of the field of sample holders and supports for the investigation of materials by X-ray diffraction. FUNDAMENTALS OF DESCRIPTION

[0002] With the recent emergence of serial crystallography (SX) in X-ray free electron lasers (XFELs), and more recently in synchrotron light sources, it is now possible to analyze the structure of biological macromolecules under conditions very close to physiological ones.

[0003] Advances in structural biology have increasingly enhanced our ability to understand the role that macromolecules play in the biological environment. This has helped to rationalize drug search and development strategies for various diseases; one example was the determination of the crystallographic structure of the main protease involved in the invasion of human cells by SARS-CoV-2, the virus responsible for the COVID-19 pandemic. These advances are largely a result of the increased number and accessibility of synchrotron light sources, particularly high-brightness third-generation sources and, more recently, fourth-generation sources, such as the Sirius® synchrotron light source (National Center for Research in Energy and Materials, Brazil).

[0004] However, in both XFEL and synchrotron light sources, the development of SX depends heavily on hardware and software systems dedicated to data processing, as well as the production and handling of crystals with the necessary quality for diffraction experiments that result in usable data sets. With respect to experimental techniques, the expansion and systematization of SX is mainly affected by the lack of standardization of sample holder systems and crystal sample preparation, as well as the incorporation of ligands or ligand fragments into the crystals. Petition 870250005562, dated 01 / 23 / 2025, page 9 / 42 2 / 19

[0005] Sampling systems can be classified into two categories: i) those that exhibit a crystal migration process, which are called flow sampling systems; and ii) those that do not exhibit a crystal migration process relative to the delivery device, which are called fixed sampling systems.

[0006] In fixed sample systems, in particular, the crystals are in a holder that is generally moved in front of the X-ray beam during the diffraction experiment. These systems are the most commonly implemented in synchrotron light experimental stations, which in many cases prove to be versatile, efficient, and with low sample consumption. Currently available fixed sample systems for protein crystallization, despite being widely used for their efficiency and low sample consumption, have some disadvantages. They limit the orientation of the crystals during data collection, make it difficult to align small or irregular crystals, and can increase susceptibility to damage caused by X-rays. In addition, manual repositioning for data collection in different regions can make the process less efficient, making a robotically movable solution preferable when feasible. STATE OF THE ART

[0007] Patent document WO2020159209 discloses a sample holder having simplified manufacture and use, designed for use in X-ray diffraction crystallography assays to visualize the crystalline structure of proteins at room temperature. It is presented as a fixed sample system, provided with a screen structure in which the sample and the crystallization solution are deposited and fixed. The screen structure is covered by polymeric films on both the upper and lower surfaces in order to seal them, preventing the evaporation of the crystallization solution. The material of the screen structure is preferably nylon, while the sealing polymeric films are preferably polyamide. Petition 870250005562, dated 01 / 23 / 2025, page 10 / 42 3 / 19

[0008] In a publication by Feiler et al., 2019 (doi: 10.3791 / 59722), the results of using commercial sample holders combined with a commercial magnetic base are revealed, facilitating the mounting of the sample holder on a goniometer for proper alignment with the X-ray source of the crystallography assay. The sample holders used are two models called XtalTool and XtalTool HT (Jena Bioscience GmbH, Germany), which have: a plastic support structure; a bio-inert polyamide film, fixed to the plastic support structure, and an amorphous cyclic olefin copolymer (COC) film, also fixed to the plastic support structure, in such a way that there is a space between the polyamide film and the COC film, in which the sample and the crystallization solution are deposited. The COC film is transparent to X-rays and the polyamide is highly transparent to X-rays.

[0010] Since the XtalTool and XtalTool HT sample holders come pre-assembled from the factory, with the polyamide and COC films fixed to the plastic support structure, sealing its internal area, to perform crystallization in the sample holder it is necessary to perforate the COC film to insert the sample and crystallization solution and then seal the COC film by applying heat and pressure to the perforated area. It should be noted that correct sealing of the COC is only possible if the perforation is relatively small and localized, requiring a precise and careful operation in the preparation of the sample holder.The XtalTool HT sample holder has the added advantage of fitting inside a standard SPINE magnetic CryoVial vial (Jena Bioscience GmbH, Germany) while mounted on the magnetic base, so that the vial provides a gripping area for manipulation by a robotic arm, allowing for automated mounting and alignment of the sample holder on the goniometer.

[0011] With regard to the state of the art, no sample holder for X-ray crystallography experiments has been found up to the time of filing of this application that combines the advantages of vapor diffusion crystallization using commercial crystallization plates and is compatible with systems. Petition 870250005562, dated 01 / 23 / 2025, page 11 / 42 4 / 19 automated crystal imaging, sealing and mounting of the device on a goniometric base, as well as the exchange of samples between in situ diffraction experiments at room temperature manually or using a robotic arm (automated sample exchange), adding to this the facilities for ligand sorting, ligand fragment sorting, serial data acquisition and experimental phasing (S-SAD), without the need for subsequent sample manipulation for each diffraction experiment preparation. BRIEF DESCRIPTION OF THE INVENTION

[0012] One of the objectives of the present invention is to provide a sample holder and a method for preparing crystal samples for X-ray diffraction crystallography experiments, especially for macromolecules, using said sample holder, which overcomes the difficulties of the prior art, presenting as advantages: crystallization in the sample holder itself, data collection from multiple crystals, ease of preparation, maintenance of sample moisture for relatively long periods in the sample holder, good sealing of the sample holder, in addition to enabling automated mounting and alignment of the sample holder in the goniometer for subsequent X-ray diffraction measurement, in situ diffraction experiments at room temperature, ligand screening, ligand fragment screening, serial data acquisition and native experimental phasing (S-SAD).

[0013] The objectives of this description are achieved by a sample holder comprising:

[0014] a disc-shaped support structure comprising: at least two projections, at least one on each side of the support structure; a central opening; at least two lateral openings; at least two channels, each channel extending from each lateral opening to at least one central opening; wherein a first film of X-ray transparent material is fixed to a first surface of the support structure, covering and sealing at least the region comprising the opening. Petition 870250005562, dated 01 / 23 / 2025, page 12 / 42 5 / 19 central, the side openings and the channels;

[0015] a sealing ring, configured to cover and seal a second surface of the supporting structure, comprising: at least two fastening elements, at least one on each of its sides, configured to fasten onto the protrusions of the supporting structure; a fastening rod configured to fit into a commercial magnetic base; and a viewing opening extending over the entire region comprising the central opening, the side openings and the channels; wherein a second film of X-ray transparent material is fixed to the surface of the sealing ring, covering at least the entire region of the viewing opening; and

[0016] an adapter, having a cylindrical body with a through hole in its main axis configured to house the fixing rod; provided with a variation of section in the lower portion of the through hole, configured to attach to the magnetic base; wherein the adapter has a support surface in its upper portion, with the shape of a double wedge with a central slot, configured to support and fix a portion of the assembly formed by the support structure and the sealing ring.

[0017] Regarding materials transparent to x-rays, examples include COC, Mylar, Mica, Graphene, and Kapton. In the present invention, the use of COC on both faces of the sample holder of the present invention is preferable, given its greater transparency as indicated in the results presented in Figure 13 and its versatility from both technical and economic points of view.

[0018] The objectives of the present description are also achieved by a method of sample preparation for crystallography experiments using the aforementioned sample holder, comprising:

[0019] to crystallize a biological sample in the central opening of the support structure;

[0020] Pipette humidification solution into the side openings of the support structure; Petition 870250005562, dated 01 / 23 / 2025, page 13 / 42 6 / 19

[0021] fit the sealing ring onto the support structure;

[0022] assemble the assembly formed by the support structure and the sealing ring onto the adapter; and

[0023] assemble the set consisting of the support structure, the sealing ring and the adapter onto the magnetic base. BRIEF DESCRIPTION OF THE FIGURES

[0024] The present invention is illustrated in the embodiments shown in Figures, as briefly described below.

[0025] Figure 1 is a schematic perspective representation of one embodiment of the sample holder described herein.

[0026] Figure 2 is a schematic perspective representation of a support structure, according to an embodiment of the sample holder of the present description.

[0027] Figure 3 is a schematic perspective representation of a sealing ring, according to an embodiment of the sample holder of the present description.

[0028] Figure 4 is a schematic perspective representation of an adapter, according to an embodiment of the sample holder of the present description.

[0029] Figure 5 is a schematic perspective representation of a commercial magnetic base for fixing to a goniometer, of the state of the art.

[0030] Figure 6 is a schematic representation of the assembly of a set comprising a support structure and a sealing ring, according to an embodiment of the sample holder of the present description.

[0031] Figure 7 is a schematic representation of a support structure for an embodiment of the sample holder described herein, in front (left), side (center) and rear (right) views, respectively.

[0032] Figure 8 contains schematic representations of a sealing ring of an embodiment of the sample holder of the present description, in views Petition 870250005562, dated 01 / 23 / 2025, page 14 / 42 7 / 19 superior (A), frontal (B), lateral (C), posterior (D) and inferior (E), respectively.

[0033] Figure 9 contains schematic representations of an adapter of an embodiment of the sample holder of the present description, in front (A') and side (B') views, and views of sections AA (C'), BB (F') and CC (G').

[0034] Figure 10 is a representation of a flow of steps of an embodiment of the sample preparation method for crystallography experiment, according to the present description.

[0035] Figure 11 shows the 3D technical drawing of the robotic arm tool (gripper) for performing automatic sample changes using the present invention, including the sample holder positioned inside the tool.

[0036] Figures 12 A and B are graphs of evaporation tests of a drop in a sealed sample holder that present the evaporation data of the sample holder system over time, measured in hours, using Polyethylene Glycol (PEG) 3350 solution at different concentrations. There are three distinct data sets, each corresponding to a different concentration of the PEG 3350 material: 5%, 10%, and 20%. The graph in Figure 12B also includes a curve fitted by a polynomial fit, indicated by the legend “Polynomial fit”.

[0037] Figure 13 shows the transparency of the combination of COC + COC, COC + Mylar, COC + Mica, COC + Kapton Tape, COC + Kapton Film and air films under X-rays.

[0038] Figure 14 provides a molecular visualization of a protein-ligand complex, showing the interaction between a ligand (enclosed in a grid) and specific amino acids (labeled) within the protein structure, co-crystallized in the sample holder of the present invention. DETAILED DESCRIPTION OF THE MODALITIES

[0039] The present description refers to a sample holder and a method of sample preparation for crystallography experiments using said sample holder.

[0040] Figure 1 shows a version of the sample holder (1) of Petition 870250005562, dated 01 / 23 / 2025, page 15 / 42 8 / 19 present description, comprising its four main elements, which are: a support structure (10), a sealing ring (20), an adapter (30) and a magnetic base (40) for fixing to a goniometer. Each of these elements can be seen individually in Figures 2 to 5.

[0041] Figure 2 illustrates an embodiment of the disc-shaped support structure (10). In this embodiment, the support structure (10) comprises: two projections (11), one on each side of the support structure (10); a central opening (12); two lateral openings (13); and two channels (14), wherein each channel (14) extends from each lateral opening (13) to the central opening (12).

[0042] The central opening (12) provides an internal environment in which the sample crystallization step takes place, as will be described below. This central opening (12) communicates fluidly with the side openings (13), through the channels (14), providing a common atmosphere to the three openings, which facilitates maintaining the sample moisture at parameters suitable for carrying out the test, for a period of 12 to 60 hours after the preparation of the sample holder.

[0043] In its assembled configuration (Figure 7), the support structure (10) has a first surface (17), configured to form a closed internal environment with the assembly of the other parts, and a second surface (18), in contact with the sealing of the sample holder (1).

[0044] Figure 3 illustrates an embodiment of the sealing ring (20), which is configured to cover and seal the first surface of the support structure (10). In the embodiment shown, the sealing ring (20) comprises: two fastening elements (21), one on each of its sides, configured to be fixed to the protrusions (11) of the support structure (10); a fastening rod (22) configured to fit into a magnetic base (40); and a viewing opening (23) that extends across the entire region corresponding to the central opening (12), the side openings (13) and the channels (14) of the support structure (10). Petition 870250005562, dated 01 / 23 / 2025, page 16 / 42 9 / 19

[0045] In one embodiment, both the support structure (10) and the sealing ring (20) are made of polymeric material.

[0046] In one embodiment, both the support structure (10) and the sealing ring (20) are produced by means of 3D printing.

[0047] Figure 4 illustrates an embodiment of the adapter (30) having a cylindrical body with a through hole (32) in its main axis configured to house the fixing rod (22). The adapter (30) is provided with a section variation (34) in the lower portion of the through hole (32), configured to attach to a locking pin present in the magnetic base (40).

[0048] The adapter (30) has a support surface (31) in its upper portion, with a double wedge shape with a central slot, configured to support and fix a lower portion of the assembly formed by the support structure (10) and the sealing ring (20).

[0049] In one embodiment, the adapter (30) is made of a flexible material, such as silicone. The flexibility of the material results in easy assembly and disassembly of the sample holder assembly (1), while at the same time securing and giving firmness to the assembly, preventing misalignment between the fixing rod (22) and the magnetic base (40) due to handling.

[0050] Figure 5 illustrates an embodiment of the magnetic base (40) that can be used in conjunction with the sample holder (1) of the present description. In this embodiment, the magnetic base is model B5 SPINE Style (MiTeGen LLC, United States of America). This base has a fitting pin (41) with a central hole for fixing the fixing rod (22). For mounting the sample holder (1), the outer part of the fitting pin (41) is coupled to the section variation region (34) of the through hole (32) present in the adapter. This coupling secures and gives firmness to the sample holder assembly (1), preventing misalignment between the fixing rod (22) and the magnetic base (40) due to handling during the sample and sample holder preparation steps.

[0051] Figure 6 is a schematic representation of the assembly of Petition 870250005562, dated 01 / 23 / 2025, page 17 / 42 10 / 19 assembly comprising support structure (10) and sealing ring (20), of one embodiment of the present description. In this representation, arrows are arranged between the elements that interface with each other, in the assembled configuration of the sample holder (1). A first film of X-ray transparent material, preferably COC (15), is fixed to the outer surface of the support structure (10), covering at least the region comprising the central opening (12), the side openings (13) and the channels (14). Similarly, a second X-ray transparent film, preferably COC (25), is fixed to the outer surface of the sealing ring (20), covering at least the entire region of the viewing opening (23).

[0052] In the embodiment represented in Figure 6, the first film (15) is fixed to a first surface (17) of the support structure (10), and the second film (25) is fixed to the sealing ring (20) by means of double-sided adhesive tapes (16,26) in the form of a ring, the double-sided adhesive tapes (16, 26) being provided with an opening in their central region, corresponding to the region of the viewing opening (23).

[0053] In one embodiment, the films (15, 25) are fixed to a first surface (17) of the support structure (10) and to a surface of the sealing ring (20) by applying fixing means selected from the group comprising curable liquid adhesive, double-sided adhesive tape (16, 26) and combinations thereof.

[0054] Figure 7 (left) is a front view of an embodiment of the support structure (10) of the sample holder (1) of the present description. In this view it is possible to observe on the first surface (17) of the support structure (10) its central opening (12), where crystallization takes place, its side openings (13), which hold liquid to maintain the internal moisture of the sample holder, and its channels (14), which connect the volume of said openings.

[0055] Figure 7 (right) is a rear view of an embodiment of the support structure (10) of the sample holder (1) of the present description. In this view it is possible to observe the second surface (18) of the support structure (10), Petition 870250005562, dated 01 / 23 / 2025, page 18 / 42 11 / 19 which is covered by the film (15) in the assembly of the sample holder (1).

[0056] Figure 8 contains schematic representations of a sealing ring (20) of an embodiment of the sample holder (1) of the present description, in top (A), front (B), side (C), rear (D) and bottom (E) views, respectively. In these figures the fastening elements (21), the fastening rod (22) and the central opening (23) are illustrated in detail.

[0057] Figure 9 contains schematic representations of an adapter (30) of an embodiment of the sample holder (1) of the present invention, in front (A') and side (B') views, respectively. In more detail, these figures indicate as follows: (i) A': front view of the adapter (30). Arrows A pass through the axis of symmetry of the part. In this view, the external shape of the part can be seen, in particular how it fits into the magnetic base (40); (ii) B': side view of the adapter (30). Arrows B pass through the axis of symmetry of the part. In this view, the external shape of the part can be seen, in particular, the fitting into the magnetic base (40) (lower arrow B) and the upper opening where the sample holder (1) is fitted into the adapter (30) (upper arrow B); (iii) C': section AA is the sectional view in the front view (A) showing the interior of the part, more particularly, the central hole of the adapter (30). Circle D indicates the fitting of the adapter (30) with the sample holder (1) and circle E indicates the fitting of the adapter (30) with the magnetic base (40). Arrows C (upper and lower) indicate that the cutting plane does not pass through the axis of symmetry of the part; (iv) D': enlargement of circle D of section AA (C'). Shows in detail the opening that holds the sample holder (1) in the upper part of the adapter (30) and, in particular, the recess that exists for fitting the extended part of the sealing ring (20); (v) E': enlargement of circle E of section AA (C'). Shows what the through hole (32) of the adapter looks like in the magnetic base fitting part (40); Petition 870250005562, dated 01 / 23 / 2025, page 19 / 42 12 / 19 (v) F': section BB is a front view of the interior of the part, where the cutting plane is indicated in the side view (B'). From this view it is possible to observe the through hole (32) of the adapter (30) and the lower part of the opening that holds the sample holder (1); (vi) G': section CC is a rear view of the interior of the part, where the cutting plane is indicated in section AA (C'). From this view it is possible to observe the recess that exists for fitting the extended part of the sealing ring (20).

[0058] Figure 10 is a representation of a flowchart of a sample preparation method for a crystallography experiment, comprising the following steps:

[0059] (S1) seal the outer surface of the support structure (10) by fixing the first film (15) to it, and seal the outer surface of the sealing ring (20) by fixing the second film (25) to it;

[0060] (S2) crystallize a biological sample in the central opening (12) of the support structure (10);

[0061] (S3) pipette humidification solution into the side openings (13) of the support structure (10);

[0062] (S4) fit the sealing ring (20) onto the support structure (10);

[0063] (S5) assemble the assembly formed by the support structure (10) and the sealing ring (20) onto the adapter (30) and then assemble the assembly formed by the support structure (10), the sealing ring (20) and the adapter (30) onto the magnetic base (40) for fixing to the goniometer;

[0064] (S6) assemble the assembly formed by the support structure (10), the sealing ring (20), the adapter (30) and the magnetic base (40) in a goniometer for alignment in crystallography testing, wherein the assembly is preferably carried out by a robotic arm.

[0065] The sample holder (1) can be stored in a 24-well plate, in a configuration of the crystallization step (S2) of the biological sample, of Petition 870250005562, dated 01 / 23 / 2025, page 20 / 42 13 / 19 in accordance with the present description.

[0066] In one embodiment, in the crystallization step (S2), samples of biological macromolecules are prepared and crystallized in the central opening (12) of the support structure (10), to be used in X-ray diffraction experiments in multiple crystals. Several methods of sample preparation and crystallization are described in the state of the art, for example, in Tenboer et al., 2014 (Science 346, 1242-1246), Beale et al., 2019 (J. Appl. Crystallogr. 52, 1385-1396), and Stohrer et al. (Acta Crystallogr. D 77). The sample holder (1) of the present description has an assembly using interlocking parts, for opening and closing the region where the sample is deposited and where crystal growth takes place, so that any of the state-of-the-art crystallization methods can be conveniently applied in the present description, not being limited to those mentioned herein.

[0067] In what follows, tests carried out on a type of sample holder described here are presented, illustrating the results and advantages achieved by it.

[0068] In one embodiment, Figure 11 is a robotic arm (gripper) for automatically changing samples from the sample holder (1) of the present invention. Applications Application 1: Evaporation test

[0069] To evaluate the evaporation rate of Polyethylene Glycol (PEG) 3350 solutions, three sample sets were prepared with concentrations of 5%, 10%, and 20% PEG 3350, as well as water (ultrapure water type 1). The samples were kept in a sample holder system, and evaporation data were measured over a 60-hour period. Measurements were taken at regular intervals, recording the percentage change in droplet volume.

[0070] The results were presented in graph form (Figures 12 Petition 870250005562, dated 01 / 23 / 2025, page 21 / 42 14 / 19 A and B), comparing the three concentrations of PEG 3350 and including a polynomial fitting curve to better represent the trend of the data. The behavior of the solutions was analyzed in terms of the stability of the evaporation rate over time. The concentration of PEG 3350 did not show a significant impact on the initial stability, with all samples showing minimal evaporation in the first 50 hours.

[0071] For the 5% PEG 3350 solution, the evaporation rate remained stable, with a reduction of only 2% of the initial volume by the end of the 60 hours. The 10% solution showed a slight acceleration in the evaporation rate after the initial 50 hours, reaching a reduction of 4% of the initial volume. Finally, the 20% concentration showed the most stable behavior, with minimal variation in evaporation until the end of the experimental period.

[0072] The results suggest that, regardless of the PEG 3350 concentration, the effect on the evaporation rate was limited, with all concentrations showing a maximum variation of 4%. This finding indicates that increasing the PEG concentration does not significantly impact the evaporation behavior under experimental conditions of up to 60 hours, which reduces the dehydration of the crystals during diffraction.

[0073] Furthermore, the rate of water evaporation over time in the graph shows stable evaporation during the 12 hours. Initially, at point zero (0 h), the rate of evaporation tends to zero, meaning that the amount of water evaporated in the closed system is minimal (Fig. 12A). In the first 6 hours, water evaporation remains extremely close to the value of 0, indicating that, in this interval, the amount of water evaporated is practically irrelevant, possibly due to an experimental environment where the humidity remained constant over time. Between 6 and 10 hours, the points still show minimal variation, but still within the 1% variation range. This suggests that there was a small loss of water through evaporation, but still within an extremely controlled range. From 10 hours onwards, Petition 870250005562, dated 01 / 23 / 2025, page 22 / 42 15 / 19 shows a slight upward trend in humidity variation, but evaporation remains close to that initially observed. Thus, we see that the rate of water evaporation over time follows a very stable linear pattern, with minimal variations over the 12-hour period, especially within the first 10 hours, indicating that the ambient sample conditions of the present invention do not significantly alter the internal evaporation rate, or that the evaporation rate of solutions, even water, is very slow and stable, being on the order of 1% over at least 12 hours. Application 2: Transparency of materials with respect to X-rays

[0074] To evaluate the X-ray transparency of the materials used as optical windows during diffraction measurements in the present invention, small-angle X-ray scattering (SAXS) measurements were performed on different combinations, which allowed direct comparison between the different materials and their respective contributions to the diffraction measurements.

[0075] According to the graph in Figure 13, among the combinations tested, the COC + COC combination showed the lowest scattering intensity throughout the q range, indicating minimal contribution to the background of the scattering measurement, which makes it ideal for diffraction experiments of crystals at room temperature.

[0076] The COC + Mylar combination showed a slight increase in scattering intensity compared to the COC + COC combination, but still maintained a relatively low contribution to the background of the images obtained by SAXS.

[0077] In comparison, the COC + Mica combination resulted in scattering behavior similar to that of COC + Mylar, but with a more noticeable increase in intensity at low q values. On the other hand, the COC + Kapton tape combination showed the highest scattering intensity among all the combinations tested, especially at low q values. Petition 870250005562, dated 01 / 23 / 2025, page 23 / 42 16 / 19 q values, suggesting that Kapton tape introduces significant interference, making it a less suitable choice for experiments requiring low scattering.

[0078] The COC + Kapton film combination also resulted in a relatively high scattering intensity, although slightly lower than that of the Kapton tape. For control purposes, the contribution of air to scattering was measured, without the presence of any window. As expected, this measurement showed the lowest scattering intensity at all q values, serving as a reference for comparison with the other materials.

[0079] These results indicate that the material of the sample holder windows significantly influences the intensity of X-ray scattering, with the COC + COC combination being the most effective in minimizing the contribution of these materials to the background of diffraction measurements during the collection of data from multiple crystals under ambient temperature conditions. Application 3: Data collection with the sample holder of the present invention.

[0080] For the structural analysis of lysozyme, trypsin, thaumatin, and proteinase K proteins, multiple crystals were collected at room temperature according to the method of the present invention. During the process, parameters and statistics related to data collection, processing, and refinement of the structure of each of these proteins were recorded and analyzed. For lysozyme, data were obtained under three distinct conditions: 1) using the experimental phasing method by native sulfur scattering, 2) after incorporation of the NAG ligand via soaking technique, and 3) by serial collection of multiple crystals also at room temperature. These conditions allowed detailed comparisons of the behavior of lysozyme in different experimental contexts. The data are presented in Table 1.

[0081] Table 1: Parameters for Collection, Processing and Structural Refinement of Proteins at Room Temperature: Lysozyme, Trypsin, Petition 870250005562, dated 01 / 23 / 2025, page 24 / 42 17 / 19 Thaumatin and Proteinase K. SW (measurements of multiple crystals with small angles, 5-10° rotation). Lysozyme (SW) Trypsin (SW) Thaumatin (SW) Proteinase k (SW) Wavelength Resolution rate 33.9 - 1.37 47.71 - 1.47 38.29 - 1.31 41.35 - 1.51 (1.419 - 1.37) (1.523 - 1.47) (1.357 - 1.31) (1.564 - 1.51) Space group P 43 21 2 P 31 2 1 P 41 21 2 P 43 21 2 Unit cell 79.41 79.41 55.09 55.09 58.66 58.66 151.6 68.42 68.42 37.49 90 90 90 109.42 90 90 90 90 90 103.81 90 90 90 Total reflections 50489 (5008) 120 141425 (14541) 1036972 (99647) 78737 (7714) Unique reflections 25600 (2535) 32771 (3212) 64036 (6316) 39374 (3857) Multiplicity 2.0 (2.0) 4.3 (4.5) 16.2 (15.8) 2.0 (2.0) Completeness (%) 99.30 (99.61) 97.82 (97.42) 99.16 (99.86) 99.91 (99.95) Mean I / sigma(I) 9.40 (0.88) 8.17 (1.64) 11.96 (0.89) 6.72 (1.08) Wilson's B Factor 19.53 2.44 18.51 9.16 R-merge 0.03269 0.2147 (0.8346) 0.105 (1.889) 0.09148 R-meas (0.6216) 0.04623 (0.879) 0.245 (0.9499) 0.1085 (1.953) (0.5977) 0.1294 (0.8453) R-pim 0.03269 0.1141 (0.4395) 0.02641 (0.4835) 0.09148 CC1 / 2 (0.6216) 0.999 (0.608) 0,967 (0.536) 0.998 (0.57) (0.5977) 0.99 (0.617) R-work 0.1965 (0.3825) 0.1903 (0.2572) 0.1774 (0.3876) 0.1655 (0.2591) R-free 0.2126 (0.3982) 0.2117 (0.2962) 0.1826 (0.4220) 0.1923 (0.3087) Protein residues 129 223 207 279 RMS (linkages) 0.003 0.003 0.006 0.007 RMS (angles) 0.67 0.70 0.86 0.90 Ramachandran 98.43 98.64 98.54 97.11 favored (%) Ramachandran 1.57 1.36 1.46 2.89 allowed (%) Outliers 0.00 0.00 0.00 0.00 Ramachandran (%) Outliers 0.00 0.57 0.00 0.00 of rotamers (%) Clashscore 3.06 2.24 2.31 0.50 Average B-factor 22.83 9.02 22.79 11.74 Macromolecules 22.00 7.05 21.48 10.05

[0082] The incorporation of the NAG (2-Acetamido-2-deoxy-beta-D-glucopyranose) ligand into lysozyme was performed by soaking. For this, the ligand was added to the droplet containing lysozyme crystals, allowing the diffusion of NAG to the protein binding site. Structural analysis revealed the interaction between NAG and specific protein residues, which were highlighted in the molecular visualization. The resulting three-dimensional structure showed the ligand, with an electron density mesh around it, confirming its position and conformation within the binding site. The secondary structure of the protein, composed of helices and beta sheets, was represented in the background, providing context for the location of the protein binding site (Figure 14). Petition 870250005562, dated 01 / 23 / 2025, page 25 / 42 18 / 19

[0083] The binding site shows the main interactions of amino acids with the NAG ligand, with the ligand well defined within the electron density and interacting directly with several important lysozyme residues, which have been labeled and displayed with the three-letter code. The collection parameters are arranged in Table 2.

[0084] Table 2: Lysozyme Collection, Processing and Structural Refinement Parameters under Different Conditions: SW-SAD (native sulfur phasing by multiple crystal measurements with small angles, 5-10° rotation), NAG Ligand (multi-crystal measurements with small angles, 5-10° rotation, with NAG ligand incorporation by soaking) and SX (serial measurement of multiple crystals, without rotation). Wavelength Lysozyme (SW_S-SAD) Lysozyme ((ligNAG) Lysozyme (SX) Resolution range 35.64 - 2.2 39.69 - 1.83 39 - 2.8 (2.9 - 2.8) (2.279 - 2.2) (1.895 - 1.83) Space group P 43 21 2 P 43 21 2 P 43 21 2 Unit cell 79.69 79.69 79.38 79.38 37.93 78 78 36 90 90 90 37.69 90 90 90 90 90 90 Total reflections 1254708 (113773) 122372 (12914) 5236 (526) Unique reflections 6539 (626) 11151 (1091) 2998 (290) Multiplicity 191.9 (181.5) 11.0 (11.8) 1.7 (1.8) Completeness (%) 99.97 (100.00) 99.86 (100.00) 99.93 (100.00) Mean I / sigma(I) 53.52 (21.29) 3.44 (1.56) 9.18 (7.89) Wilson's B-factor 13.24 10.85 27.32 R-merge 0.1524 (0.3833) 0.5471 (1.296) 0.06697 (0.07231) R-meas 0.1528 (0.3844) 0.5744 (1.354) 0.0947 (0.1023) R-pim 0.01095 (0.02856) 0.1698 (0.3833) 0.06697 (0.07231) R-split 0.492(0.423) CC1 / 2 0.999 (0.994) 0.937 (0.702) 0.978 (0.977) R-work 0.1781 (0.2151) 0.2930 (0.3498) 0.2021 (0.2300) R-free 0.2354 (0.2308) 0.3352 (0.4591) 0.2486 (0,2014) Protein residues 129 129 129 RMS (linkages) 0.002 0.005 0.005 RMS (angles) 0.50 0.87 0.67 Ramachandran 98.43 95.28 95.16 favored (%) Ramachandran 1.57 3.94 4.84 allowed (%) Outliers 0.00 0.79 0.00 Ramachandran (%) Rotamer outliers (%) 0.95 0.00 0.80 Clashscore 3.05 18.57 9.46 Average B factor 16.09 12.55 21.87 Macromolecules 12.39 21.87 Petition 870250005562, dated 01 / 23 / 2025, page 26 / 42 19 / 19

[0085] Although embodiments of the processes and products described have been presented in this report, the scope of protection is not intended to be limited to the literal wording thereof. Therefore, the description should be interpreted not as restrictive, but merely as examples of particular embodiments that retain the inventive concept presented herein. A person skilled in the art may readily apply the teachings presented herein to analogous solutions arising therefrom, limited only by the scope of the claims in this application.

Claims

CLAIMS 1. Sample holder characterized by comprising: a disc-shaped support structure (10), comprising: at least two projections (11), at least one on each side of the support structure (10); a central opening (12); at least two lateral openings (13); at least two channels (14), each channel (14) extending from each lateral opening (13) to at least one central opening (12); wherein a first film of X-ray transparent material (15) is fixed to a first surface of the support structure (10), covering and sealing at least the region comprising the central opening (12), the lateral openings (13) and the channels (14);a sealing ring (20), configured to cover and seal a second surface of the support structure (10), the sealing ring (20) comprising: at least two fastening elements (21), at least one on each of its sides, configured to fasten to the protrusions (11) of the support structure (10); a fastening rod (22) configured to fit into a commercial magnetic base (40); and a viewing opening (23) extending over the entire region comprising the central opening (12), the side openings (13) and the channels (14); wherein a second film of X-ray transparent material (25) is fixed to the surface of the sealing ring (20), covering at least the entire region of the viewing opening (23); and an adapter (30), having a cylindrical body with a through hole (32) in its main axis configured to house the fastening rod (22);provided with a section variation (34) in the lower portion of the through hole (32), configured to attach to the magnetic base (40); wherein the adapter (30) has a support surface (31) in its upper portion configured to support and fix a portion of the assembly formed by the support structure (10) and the sealing ring (20).

2. Sample holder, according to claim 1, characterized in that the support surface (31) has a double wedge shape with a central slot. Petition 870250005562, dated 23 / 01 / 2025, page 28 / 42 2 / 3 3. Sample holder, according to claim 1, characterized in that the films (15, 25) are fixed to the support structure (10) and the sealing ring (20) by means of fixing means (16, 26) selected from the group comprising curable liquid adhesive, double-sided adhesive tape and combinations thereof.

4. Sample holders, according to any one of claims 1 to 3, characterized in that the films of x-ray transparent materials are selected from the COC, Mylar, Mica and Kapton groups.

5. Sample holders, according to claim 4, characterized in that the films of x-ray transparent materials are preferably COC.

6. Sample holder, according to any one of claims 1 to 5, characterized by the fact that it collects data from multiple crystals.

7. Sample holder, according to any one of claims 1 to 6, characterized in that the sealing and assembly of said sample holder is on a goniometric basis.

8. Sample holder, according to any one of claims 1 to 7, characterized in that the exchange of samples is carried out manually or using a robotic arm.

9. Sample preparation method using the sample holder as defined in any one of claims 1 to 8, characterized by comprising: (S1) sealing the outer surface of the support structure (10) by fixing the first film (15) thereto, and sealing the outer surface of the sealing ring (20) by fixing the second film (25) thereto; (S2) crystallizing a biological sample in the central opening (12) of the support structure (10); (S3) pipetting humidifying solution into the side openings (13) of the support structure (10); Petition 870250005562, dated 23 / 01 / 2025, p.29 / 42 3 / 3 (S4) fit the sealing ring (20) onto the support structure (10); (S5) assemble the assembly formed by the support structure (10) and the sealing ring (20) onto the adapter (30) and then assemble the assembly formed by the support structure (10), the sealing ring (20) and the adapter (30) onto the magnetic base (40) for fixing to a goniometer; (S6) assemble the sample holder assembly (1), formed by the support structure (10), the sealing ring (20), the adapter (30) and the magnetic base (40), onto a goniometer.

10. Method according to claim 9, characterized in that step (S6) is performed manually or by a robotic arm.

11. Method, according to any one of claims 9 to 10, characterized by the fact that it collects data from multiple crystals.

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