A crystallization plate suitable for in situ x-ray diffraction of biomolecules

Abstract

This invention provides a crystallization plate suitable for in-situ X-ray diffraction of biomolecules, comprising: a body and multiple crystallization units formed within the body; each crystallization unit includes a through-hole and a frustum-shaped sample slot support within the through-hole; the lower bottom surface of the frustum-shaped sample slot support is connected to the lower bottom surface of the through-hole, and the upper bottom surface of the frustum-shaped sample slot support is located between the upper top surface and the lower bottom surface of the through-hole; the side surface of the frustum-shaped sample slot support and the side surface of the through-hole form a crystallization buffer bath. The crystallization plate of this invention is applicable to in-situ testing of biomolecular crystals, and features simple operation, good compatibility, high efficiency, and low cost. It can greatly facilitate the rapid collection of single-crystal diffraction data or drug screening at synchrotron radiation crystallography beamlines under room temperature in-situ conditions.

Description

Technical Field

[0001] This invention relates to the field of protein crystal structure analysis, and in particular to a crystallization plate suitable for in-situ X-ray diffraction of biomolecules. Background Technology

[0002] The resolution of protein crystal structures plays a crucial role in exploring the functions of various proteins in living organisms and studying the origin of life. As of June 26, 2022, the Rectified Protein Structure Database (RCSB) had resolved the structures of 191,869 proteins, with over 80% of these structures still determined using X-ray diffraction (XRD). This demonstrates that XRD is the primary method for obtaining the structures of biological macromolecules. In recent years, with upgrades in detector technology and the use of synchrotron radiation sources, the data collection efficiency of biological macromolecular crystals has significantly improved, increasing the amount of diffraction data collected from 2-3 sets per hour to over 20 sets per hour. This has paved the way for the application of high-throughput protein crystallography in drug discovery. Developing screening platforms for drug development based on synchrotron radiation crystallography beamlines has become a cutting-edge international trend.

[0003] With the rapid development of structural biology, fragment-based drug discovery has received increasing attention and is one of the important methods for lead compound development. Structural biology methods can provide three-dimensional information about the interaction between fragment compounds and target proteins, which is the foundation for structure-based drug molecule optimization. Structural information can not only greatly accelerate the drug development process but also significantly reduce the cost of new drug development. The use of biomacromolecule structures for functional studies and lead compound and antibody drug screening is receiving increasing attention from the scientific and industrial communities. Therefore, more and more researchers are investing more time and effort in the development and application of in-situ techniques. In-situ techniques involve setting up crystallization and testing in the same location. This method not only simplifies the structural analysis process and reduces the additional costs in the testing process but also accelerates testing efficiency. In recent years, related research has proposed the concept of in-situ plates. Among them, a domestic patent document (application number 202111259671.6) discloses an in-situ crystallization device. Its in-situ main plate can be used as a lipid cubic phase (LCP) plate, a sitting drop plate, and a hanging drop plate, and can simultaneously accommodate multiple in-situ sample plates. It can be used directly for LCP crystallization experiments, assembled with a sitting drop in-situ crystallization plate for sitting drop crystallization experiments, and assembled with a hanging drop in-situ crystallization plate for hanging drop crystallization experiments. However, this device design is relatively complex, and the crystallization plate absorbs a large amount of X-rays, producing severe background scattering. Furthermore, X-rays scattered from the sample are easily blocked by the crystallization plate, affecting subsequent experimental results. Currently available crystallization plates generally suffer from time-consuming retrieval and sample freezing damage, while the testing process consumes a significant amount of manpower and resources.

[0004] Therefore, how to solve the problems of complex design and severe background scattering during use of existing crystal plates has become one of the urgent problems to be solved by those skilled in the art.

[0005] It should be noted that the above introduction to the technical background is only for the purpose of providing a clear and complete explanation of the technical solutions of this application and facilitating understanding by those skilled in the art. It should not be assumed that these technical solutions are known to those skilled in the art simply because they have been described in the background section of this application. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a crystallization plate suitable for in-situ X-ray diffraction of biomolecules, which solves the problems of complex crystallization plate design and severe background scattering during use in the prior art.

[0007] To achieve the above and other related objectives, the present invention provides a crystallization plate suitable for in-situ X-ray diffraction of biomolecules, comprising: a body and a plurality of crystallization units formed in the body;

[0008] The crystallization unit includes a through hole and a frustum-shaped sample slot support inside the through hole;

[0009] The lower bottom surface of the frustum-shaped sample slot support is connected to the lower bottom surface of the through hole, and the upper bottom surface of the frustum-shaped sample slot support is located between the upper top surface and the lower bottom surface of the through hole.

[0010] The side of the frustum-shaped sample holder and the side of the through hole form a crystallization buffer tank.

[0011] Optionally, the frustum-shaped sample slot support is a regular frustum-shaped sample slot support.

[0012] Optionally, the frustum-shaped sample slot support is a hollow support that extends vertically.

[0013] Alternatively, a loading film may be provided on the upper bottom surface of the frustum-shaped sample holder.

[0014] Alternatively, the loading film may be a polyimide film or a cyclic olefin copolymer film.

[0015] Optionally, the through hole is a straight hole structure.

[0016] Alternatively, the plurality of said crystallization units are arranged in an array.

[0017] Alternatively, the distance between the axial center lines of any two adjacent crystallization units is 5 mm to 15 mm.

[0018] As described above, the crystallization plate of the present invention has the following beneficial effects:

[0019] 1. The crystallization plate of the present invention can directly place the crystal on the X-ray path to achieve in-situ diffraction data acquisition.

[0020] 2. The frustum-shaped sample slot support of the crystallization unit in the crystallization plate of the present invention is a hollow support that runs through the top and bottom, which ensures that X-rays can directly irradiate the sample and avoids the influence of the crystallization plate on X-rays.

[0021] 3. The crystallization plate of the present invention can be used directly for drop crystallization experiments and in-situ diffraction data acquisition experiments, eliminating the cumbersome step of retrieving the crystal from the crystallization plate in conventional methods.

[0022] 4. The crystallization buffer tank of the crystallization unit in the crystallization plate of the present invention is characterized by being wider at the top and narrower at the bottom, which can effectively realize in-situ diffraction experiments and data acquisition during large-angle rotation. During in-situ diffraction, X-rays are not interfered with by the crystallization plate (X-rays scattered by the sample are not blocked by the crystallization plate and will not affect subsequent experimental results), thus providing a stable and efficient sample loading technique for in-situ crystal diffraction experiments in structural biology, and greatly facilitating in-situ diffraction experiments based on crystallization plates in synchrotron radiation. Attached Figure Description

[0023] Figure 1 The diagram shown is a top view of the crystallization plate of the present invention.

[0024] Figure 2 The diagram shown is a bottom view of the crystallization plate of the present invention.

[0025] Figure 3 The diagram shown is a cross-sectional view of the crystallization plate of the present invention at L'-L.

[0026] Figure 4 The diagram shown is a schematic representation of the crystallization plate of the present invention under X-ray diffraction.

[0027] Component designation explanation

[0028] 1. Crystallization plate

[0029] 11 body

[0030] 2 Crystallization Unit

[0031] 21. Frustum-shaped sample holder

[0032] 22 Crystallization buffer tank

[0033] 3. Loading the film

[0034] Steps S1 to S3 Detailed Implementation

[0035] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0036] Please see Figures 1-4 It should be noted that the illustrations provided in this embodiment are only schematic representations of the basic concept of the present invention. Therefore, the illustrations only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0037] Example 1

[0038] like Figures 1-3 As shown, this embodiment provides a crystallization plate 1, which includes a body 11 and a plurality of crystallization units 2 formed in the body 11.

[0039] Specifically, the crystallizing plate 1 is a rectangular crystallizing plate. The crystallizing plate 1 can have a regular or irregular structure, and the structure can be set according to actual needs, without limitation here. As an example, the outer shell of the body 11 is relatively flat, which can well conform to the installation and use of the diffractometer and meet the usage requirements.

[0040] like Figure 1 and Figure 3 As shown, the crystallization unit 2 includes a through hole and a frustum-shaped sample holder 21 within the through hole. As an example, the through hole has a sidewall, the height of which is greater than the thickness of the crystal body 11; the sidewall of the through hole is connected to the surface of the body 11.

[0041] Specifically, as an example, the through hole is a straight hole structure, and the sidewall of the through hole is perpendicular to the body 11 of the crystal plate 1. As an example, the through hole can also be other hollow three-dimensional structures, such as a cube or a hexagon, or other irregular hollow three-dimensional structures, which are not limited here.

[0042] like Figure 3As shown, each of the through holes is provided with a frustum-shaped sample slot support 21; as an example, the frustum-shaped sample slot support 21 is a hollow support that extends vertically (only the side of the frustum-shaped sample slot support is retained). As an example, the frustum-shaped sample slot support 21 is a regular truncated pyramidal sample slot support, and the straight line connecting the center points of the upper and lower base surfaces of the frustum-shaped sample slot support 21 is perpendicular to the lower base surface. The frustum-shaped sample slot support 21 in the crystallization plate 1 of the present invention is a hollow support, which ensures that X-rays can directly irradiate the sample and avoids the influence of the crystallization plate 1 on X-rays.

[0043] Specifically, such as Figure 3 As shown, a loading film 3 is laid on the upper bottom surface of the frustum-shaped sample holder 21 for loading the sample; as an example, the loading film 3 is a polyimide film or a cyclic olefin copolymer film, and the loading film 3 is a polymer material film with a low X-ray scattering signal intensity during X-ray diffraction at a wavelength of 1 angstrom. The polymer material has little influence on the XRD (X-Ray Diffraction) peak of the analyte and has good blocking performance.

[0044] Specifically, such as Figure 3 As shown, the lower bottom surface of the frustum-shaped sample holder 21 is connected to the lower bottom surface of the through hole, and the upper bottom surface of the frustum-shaped sample holder 21 is located between the upper top surface and the lower bottom surface of the through hole. As an example, the area of ​​the lower bottom surface of the frustum-shaped sample holder 21 is equal to the area of ​​the lower bottom surface of the through hole, and the lower bottom surface of the frustum-shaped sample holder 21 is connected to the lower bottom surface of the through hole; the height of the frustum-shaped sample holder 21 is less than the height of the through hole, thus the side surface of the frustum-shaped sample holder 21 and the side surface of the through hole form a crystallization buffer tank 22. The crystallization buffer tank 22 in the crystallization plate 1 of the present invention is characterized by being wider at the top and narrower at the bottom, which can effectively realize in-situ diffraction experiments during large-angle rotation; at the same time, it avoids leakage of the crystallization buffer during the rotation of the crystallization plate 1 (avoiding the droplet flow, strong background diffraction, and the obstruction of the liquid hole edge affecting the diffraction angle caused by using ordinary crystallization plates, improving the quality of crystal diffraction, increasing the angle at which data can be collected from a single crystal, and improving the completeness of diffraction data). When the crystallization plate 1 of the present invention is used, the bio-crystallized macromolecules are carried on the loading film 3, and the crystallization solution is placed in the crystallization buffer tank 22, thereby separating the bio-macromolecules from the crystallization buffer solution, while ensuring that the bio-macromolecules on the sample stage can be crystallized through vapor diffusion.

[0045] like Figure 1 The image shown is a top view of the crystallizing plate 1. Figure 2The image shows a bottom view of the crystallizing plate 1. As an example, multiple crystallizing units 2 are arranged in an array; in this embodiment, the array of crystallizing units 2 is 12 rows and 8 columns. It should be noted that the multiple crystallizing units 2 can also be arranged in other shapes, such as circles, triangles, etc., which will not be elaborated here. As an example, the distance between the axial center lines of any two adjacent crystallizing units 2 is 5mm to 15mm; in this embodiment, the distance between the axial center lines of any two adjacent crystallizing units 2 is 9mm; the distance between the axial center lines of adjacent crystallizing units 2 can be set as needed and is not limited here.

[0046] In one implementation of the present invention, when the crystallization plate 1 is applied to the field of protein crystal structure analysis, the loading film 3 acts as a drop-sitting platform, and the crystallization plate 1 is applied to drop-sitting crystallization experiments or in-situ diffraction data acquisition experiments. As an example, when the crystallization plate 1 is applied to in-situ diffraction data acquisition experiments, such as... Figure 4 As shown, the crystal plate 1 is mounted on the angle measuring head of a diffractometer with crystal plate clamping function. The bottom surface of the frustum-shaped sample slot support 21 faces the X-ray emission path. With the help of the rotation of the angle measuring head, in-situ diffraction data collection experiments are carried out.

[0047] like Figure 3 As shown, the bottom surface of the crystallization unit in the crystallization plate of the present invention forms a hollow frustum-shaped groove, which allows the X-ray beam path direction to be perpendicular to the surface of the crystallization plate and to pass through the hollow frustum-shaped groove on the bottom surface of the crystallization unit, ensuring that the X-rays irradiate the crystal.

[0048] The crystallization plate of this invention enables the collection of high-quality diffraction data from a single crystal with large-angle rotation, ensuring the integrity and quality of the data, thereby effectively realizing in-situ diffraction experiments based on the crystallization plate. The crystallization plate of this invention does not require storage in liquid nitrogen or cryopreservation during crystal crystallization, enabling in-situ data collection or drug screening of a single crystal under room temperature conditions; it avoids damage to the sample after cryogenic freezing, which could lead to changes in crystal structure conformation or loss of crystal diffraction ability.

[0049] The crystallization plate of this invention can be used for drop crystallization experiments and in-situ diffraction data acquisition experiments, eliminating the cumbersome step of retrieving the crystal from the crystallization plate in conventional methods.

[0050] The crystal plate of this invention enables diffraction data acquisition experiments within a large angular rotation range, and X-rays are not affected by the crystal plate, thus providing a stable and efficient sample loading technique for in-situ crystal diffraction experiments in structural biology, and greatly facilitating in-situ diffraction experiments based on crystal plates in synchrotron radiation.

[0051] The crystallization plate of this invention is used for in-situ testing of biological macromolecular crystals. It is simple to operate, has good compatibility, high efficiency and low price. It can greatly facilitate the rapid collection of single crystal diffraction data or drug screening at room temperature in situ at synchrotron radiation crystallography beamlines.

[0052] Example 2

[0053] This embodiment provides a method for a sitting-drop crystallization experiment, based on the crystallization plate described in Embodiment 1. The sitting-drop crystallization experiment method includes the following steps:

[0054] S1: Place the crystallization plate 1 on the experimental table, and lay a loading film 3 on the upper bottom surface of the frustum-shaped sample slot support 21.

[0055] Specifically, in a room temperature environment, the crystallization plate 1 is placed on the experimental stage with the upper surface of the crystallization unit 2 facing upwards, and the loading film 3 is laid on the upper bottom surface of the frustum-shaped sample holder 21 (the loading film 3 seals the upper bottom surface of the frustum-shaped sample holder 21). As an example, the loading film 3 is a polyimide film or a cyclic olefin copolymer film. The loading film 3 is a polymeric material film with a low X-ray scattering signal intensity during X-ray diffraction at a wavelength of 1 angstrom. The polymeric material has little influence on the XRD (X-Ray Diffraction) peaks of the analyte and has good blocking performance.

[0056] S2: Add biomacromolecules to the loading membrane 3 and add crystallization buffer to the crystallization buffer tank 22 to culture and crystallize.

[0057] Specifically, biomolecules are added to the loading membrane 3 using a liquid spotting machine, and then crystallization buffer is added to the crystallization buffer tank 22 for culturing and crystallization.

[0058] Based on the protein crystal obtained in step S2, an in-situ diffraction experiment can also be performed. The in-situ diffraction experiment further includes step S3 in addition to steps S1 and S2.

[0059] S3: Place the crystallization plate containing the protein crystal in the diffractometer and perform in-situ diffraction on the crystal. X-rays are incident from the bottom surface of the frustum-shaped sample holder 21; then collect diffraction data.

[0060] Specifically, a crystallization plate containing protein crystals is mounted on the measuring head of a diffractometer with a crystallization plate clamping function. The bottom surface of the frustum-shaped sample slot support 21 faces the X-ray emission path. In-situ diffraction data collection experiments are conducted by rotating the measuring head.

[0061] In summary, this invention provides a crystallization plate suitable for in-situ X-ray diffraction of biomolecules, comprising: a body and multiple crystallization units formed within the body; each crystallization unit includes a through-hole and a frustum-shaped sample slot support within the through-hole; the lower bottom surface of the frustum-shaped sample slot support is connected to the lower bottom surface of the through-hole, and the upper bottom surface of the frustum-shaped sample slot support is located between the upper top surface and the lower bottom surface of the through-hole; the side surface of the frustum-shaped sample slot support and the side surface of the through-hole form a crystallization buffer tank. The crystallization plate of this invention can achieve the collection of high-quality diffraction data from a single crystal with large-angle rotation, ensuring the integrity and quality of the data, thereby effectively realizing in-situ diffraction experiments based on the crystallization plate, and achieving in-situ data collection or drug screening of a single crystal under room temperature in-situ conditions; the crystallization plate of this invention is applied to in-situ testing of biomolecular crystals, and has the characteristics of being simple to operate, having good compatibility, high efficiency, and low cost, which can greatly facilitate the rapid collection of single-crystal diffraction data or drug screening at synchrotron radiation crystallography beamlines under room temperature in-situ conditions. Therefore, this invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0062] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A crystallization plate suitable for in situ X-ray diffraction of biomolecules, characterized in that, The crystallization plate comprises a body and a plurality of crystallization units formed in the body; The crystallization unit comprises a through hole and a frustum-shaped sample groove support in the through hole; The lower bottom surface of the frustum-shaped sample groove support is connected with the lower bottom surface of the through hole, and the upper bottom surface of the frustum-shaped sample groove support is between the upper top surface and the lower bottom surface of the through hole; The through hole is a straight hole structure, the frustum-shaped sample groove support is a right prism frustum-shaped sample groove support, and the side surface of the frustum-shaped sample groove support and the side surface of the through hole form a crystallization buffer groove which is wide at the top and narrow at the bottom; The upper bottom surface of the frustum-shaped sample groove support is provided with a loading film; The frustum-shaped sample groove support is a hollow support which penetrates from top to bottom.

2. The crystallization plate of claim 1, wherein: The loading film is a polyimide film or a cyclic olefin copolymer film.

3. The crystallization plate of claim 1, wherein: The plurality of crystallization units are arranged in an array.

4. The crystallization plate of claim 3, wherein: The spacing between the axial center lines of any two adjacent crystallization units is 5 mm to 15 mm.

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