CN110361407B - Device for protein crystal in-situ X-ray diffraction - Google Patents

Abstract

The invention discloses a device for in-situ X-ray diffraction of protein crystals, which is used for solving the problems of difficulty in picking, identifying and locating and diffracting tiny protein crystals in the field of biotechnology, and improves the utilization rate of crystals. The device consists of a two-dimensional translation and one-dimensional rotation inspection and positioning mechanism, a disc-shaped crystal box mechanism, an illumination mechanism, a microscopic imaging mechanism, a diffraction absorption mechanism and a support mechanism. The inspection and positioning mechanism implements two-dimensional translational positioning for the disc-shaped crystallizing box, and the inspection motor drives the crystallizing box to rotate to realize the inspection of the concentrically distributed crystallizing cell array on it, and there is no need to turn the crystallizing box in the middle; use the epi- or transmission of the lighting mechanism Illumination and light of different colors, find protein crystals through a long focal length microscope, and make X-rays accurately pass through the crystals with the fine adjustment of the inspection and positioning mechanism; The mouth can be automatically selected and replaced according to the size of the protein crystals.

Description

A device for in situ X-ray diffraction of protein crystals

technical field

The invention discloses a device that can install a protein crystal together with a crystallization box on an X-ray diffractometer to implement in-situ diffraction, and belongs to the field of biotechnology.

Background technique

Life science is the most challenging science today. Genome and proteome research provides infinite possibilities for the development of almost omnipotent biotechnology products, the human dream of longevity, the early diagnosis and treatment of major diseases, and the killing of bacteria and viruses. Or transformation and domestication, it is no longer a false fantasy. The development of these biotechnologies requires fine atomic structure information about protein molecular machines, because proteins are not only the structural substances of organisms, but also the executors of life activities, which are exquisite molecular machines.

The structure of protein molecular machines is very complex, consisting of thousands or more of carbon, hydrogen, oxygen, nitrogen, etc. atoms. There are four main methods to determine the atomic-level structure of protein molecular machines: crystal X-ray diffraction, nuclear magnetic resonance, cryogenic transmission electron microscopy and theoretical calculations. Crystal X-ray diffraction method is the most commonly used and most effective method at present, but the premise is to conduct protein crystallization and obtain high-quality single crystals. Because protein molecules contain many atoms, large structural flexibility, and a large amount of bound water and free water, etc. For this reason, it is particularly difficult to grow high-quality single crystals on the millimeter scale. For this reason, people have developed various methods such as vapor phase diffusion crystallization, liquid-liquid diffusion crystallization, liquid-dosing crystallization, dialysis crystallization, etc., and even send proteins to space in order to use microgravity to suppress convection in the protein solution to improve protein crystals. Growth size and quality. Among these methods, the most commonly used methods are vapor phase crystallization and liquid-liquid crystallization. Different proteins sometimes require different methods.

With the development of science and technology, the power and quality of X-rays have been greatly improved. For example, the use of synchrotron radiation and free electron laser light sources has reduced the requirements for protein crystals from millimeters to micrometers. Liquid nitrogen freezing and other operations can easily destroy the crystals, so that all previous efforts are lost.

Therefore, the main manufacturer of laboratory-level X-ray diffraction equipment, Rigaku Corporation of Japan, proposed the technology of directly installing the porous crystal plate on the X-ray diffractometer to implement diffraction, and developed a crystal plate fixing and adjustment mechanism, which can Do not use micron-thick nylon wire loops to pick up crystals for immersion and liquid nitrogen freezing. However, their technology can only illuminate micron-scale crystals by epigraphy, and the effect is not good, and it is difficult to find crystals and position them; X-ray absorption nozzles of different specifications need to be manually replaced according to the size of crystals, and X-rays need to be turned off; there is currently no batch on the market. A special crystallized plate that can be placed sideways. The core of the technical solution described in the published invention patent "In-situ Diffraction Device and Diffraction Method for Protein Crystals" (Publication No. CN108593689A) is the crystallization box for in-situ diffraction. -Technical description of ray diffractometer matching; the technical solution of sticking polymer film on both sides of the double-sided tape used in the crystallization box cannot fix crystals when the box is used on the side, and it is difficult to implement micro-nano crystals, and the double-sided tape is difficult to crystallize many proteins. The solution has problems of physical and chemical compatibility; in addition, this technical solution does not describe the layout of the protein solution and its equilibrium solution/precipitation solution, that is, it does not specify the crystallization method used, such as vapor phase diffusion, liquid- Liquid diffusion or liquid dispensing method, etc. The disclosed invention "diffraction method of biological macromolecular crystals in near-physiological state" (publication number CN108732193A) does not provide a schematic diagram of the technical implementation principle, and the content is similar to CN108593689A. The "near-physiological state" of molecular coexistence. The disclosed invention "A Serial Crystallographic Sample Transport Device and Method" (Publication No. CN109490343A) describes a motorized rotating circular protein crystal transport box, which has an annular groove inside which can hold crystals and conduct diffraction. The technology Rather than an in situ diffraction technique, it is still necessary to transfer the protein crystals from the growth solution into the box; no other companion technique is involved in this invention.

With a normal crystallizing plate, the crystals will move and the equilibration and protein fluids will flow, all of which cause diffraction experiments to fail. In addition, when using the existing commercial crystallographic plate, because its width exceeds the allowable size of the current X-ray diffractometer, the crystallizing plate needs to be diffracted twice after it is installed, and the crystallizing plate needs to be turned up and down in the middle.

Therefore, the development of new devices and crystallization plates/boxes has become the key to solving the above problems.

SUMMARY OF THE INVENTION

In order to solve the above problems and deficiencies, the present patent invents a device for in-situ X-ray diffraction of protein crystals, which can inspect and locate protein crystals in the crystallization chamber. The device consists of a disc-shaped crystallizing box, a three-degree-of-freedom inspection and positioning mechanism with two-dimensional translation and one-dimensional rotation, an illumination mechanism, a microscopic observation and imaging mechanism, an X-ray absorption mechanism and a connection support mechanism. The inspection and positioning mechanism manipulates the disc-shaped crystallizing box to realize two-dimensional translation positioning, and the inspection motor drives the crystallizing box to rotate for inspection. The fine positioning adjustment of the mechanism makes the X-rays irradiate the crystal accurately; the X-rays traveling straight after passing through the crystal are absorbed by the absorption mechanism. Among them, the disc-shaped crystallization box does not need to be installed sideways on the device when growing protein crystals in the early stage, and is usually placed horizontally in a constant temperature incubator. , equilibration solution and protein crystals are fixed so that their flow or movement can be prevented from affecting diffraction experiments.

The invention is suitable for directly fixing the crystallizing box after the crystal growth has been completed, and performing two-dimensional translation positioning of the crystallizing box in the vertical plane by means of two mutually perpendicular slide rails; With a good crystallizing cell, all the crystallizing cells on the same circumference can be diffracted through the rotation of the crystallizing box, avoiding the trouble that some crystallizing boxes need to be turned over in the half-diffraction process; The illumination light source makes the observation, identification and positioning of protein crystals more convenient and efficient; X-ray absorption nozzles of different specifications and several transmission light sources are installed on the same rack, which can be automatically selected and replaced according to the size of protein crystals, thus improving the accuracy of diffraction data. Quality; in the crystallization tank of the crystallization chamber, by using gelatinized protein solution and balance solution, porous materials to absorb the balance solution, and adsorption layer to fix crystals, they can be effectively fixed to avoid interfering with diffraction experiments. In addition, the use of this innovative invention can avoid the risk of damage to the crystals due to complex operations such as fishing, liquid nitrogen freezing, transfer, and liquid nitrogen storage for tiny protein crystals. , time cost and labor cost, the development and application of the device will have considerable economic and social benefits.

Description of drawings

1 is a schematic diagram of the structure of the device of the present invention;

Fig. 2 is A-A sectional structure schematic diagram in Fig. 1;

Fig. 3 is a schematic diagram of the first design scheme of the composite stent;

Fig. 4 is the schematic diagram of the second design scheme of the composite support;

Figure 5 is a schematic diagram of the layout of the microscope seat components;

6 is a schematic diagram of the orthographic structure of the crystal box;

7 is an orthographic view of the working positioning of the crystallization box and the composite support;

Figure 8 is a side projection view of the working positioning of the crystallization box and the composite support.

Detailed ways

In order to enable those skilled in the art to better understand the present invention, the technical solutions of the present invention are further described below with reference to the accompanying drawings and embodiments.

1. Composition

The device is composed of an inspection and positioning mechanism, a microscopic imaging mechanism, a crystal box mechanism, an illumination mechanism, a diffraction absorption mechanism and a support mechanism. Figure 1 is a preferred example. The inspection and positioning mechanism consists of a large gear 6, a toothed belt 7, a base 8, an inspection motor 9, a cantilever 10, a pinion 11, an X slide rail 12, a Y slide rail 13, an X motor 14, a Y motor 15, and a gear shaft. 16; the microscopic imaging mechanism is shown in Figure 2, which is composed of a microscope 26 and a regulator 27; the crystallizing box mechanism is shown in Figures 6, 7 and 8, consisting of a crystallizing box 5, a crystallizing tank 31, a balance liquid 32, The protein solution 33 and the protein crystal 34 are composed; the lighting mechanism is shown in Figure 2 and Figure 3, the transmission lighting source is composed of the G lamp 19, the B lamp 21 and the R lamp 23, and the blue light 35, The green light 36 emitted by the green light 29, the red light 37 emitted by the red light 30 and the white light 38 emitted by the microscope 26 form an epi-illumination light source. and protein crystal 34; the diffraction absorption mechanism is shown in Figure 2, Figure 3 and Figure 8, consisting of a composite bracket 3, a lamp nozzle motor 17, an S absorption nozzle 18, an M absorption nozzle 20, an L absorption nozzle 22, and an X-ray 25. ; The support mechanism is shown in Figure 2, which consists of a base plate 1, a base 2, and a microscope seat 4.

2. Working principle

Before installing the crystallization box 5 on the gear shaft 16 and starting the whole device to perform in-situ X-ray diffraction, a key preparatory work needs to be done, that is, growing the protein crystal 34, adding the equilibration solution 32 and the protein solution 33. In the crystallization tank 31 on the crystallization box 5, after sealing, place the crystallization box 5 in a constant temperature incubator. After the expected crystal growth is completed, take out the crystallization box 5 and install it on the gear shaft 16 sideways. How to avoid the movement of the equilibration solution 32, the protein solution 33 and the protein crystal 34, please refer to the above-mentioned "Summary of the Invention" section.

The X motor 14 and the Y motor 15 drive the X slide rail 12 and the Y slide rail 13 respectively, and cooperate with the inspection motor 9 to drive the rotation of the crystallizing box 5 through the pinion 11, the toothed belt 7 and the large gear 6, so that a specified crystallizing tank 31 The protein liquid 33 in the middle is brought into the field of view of the microscope 26, and after finding an object that may be the protein crystal 34, the illumination can be changed for further determination.

Further fine-tune the inspection and positioning mechanism so that the protein crystal 34 is just under the irradiation of the X-ray 25 . On the other hand, adjust the diffraction absorption mechanism, and select a suitable absorption nozzle according to the size of the protein crystal 34 , such as the S absorption nozzle 18 .

After the experiment of one pond is completed, the crystallization box 5 is driven by the inspection motor 9 to inspect other crystallization ponds located on the same circumferential position on the crystallization box 5 . After the experiment of the pools on the same circumference is completed, the crystallization pools on the other circumferences can be brought into the field of view of the microscope 26 using only the Y slide. And so on until it's all done.

Some specific details are described below regarding the structure of each component.

3. The structure of each component

The base plate 1 provides a mounting platform for other institutions. It is fixed on the work surface of the X-ray diffractometer by bolts. M6 and φ6 holes are machined on it, which are distributed in an array, and the central moment of adjacent holes is 25mm. Metal material is used, preferably duralumin. Bottom plate 1 is 200-500mm long and 150-300mm wide.

The base 2 is used to support the diffraction absorption mechanism and part of the lighting mechanism, such as the composite bracket 3 and the lamp nozzle motor 17, which can be in the shape of a simple cuboid, and the upper part can also be designed as a semi-cylindrical surface. The size of base 2 is: length 100-200mm, width 50-100mm, height 200-400mm. Metal material, preferably duralumin.

The composite bracket 3 is used to install multi-color lighting lamps and absorption nozzles of different specifications. Its preferred structure is shown in Figure 3. The six arms are equally distributed. Considering the rigidity of the bracket, it can be improved to the structure shown in Figure 4. The ring connects the ends of the six arms together; the number of arms is not limited to six, but can be less or more, such as four or eight. In this embodiment, the S absorption nozzle 18, the G lamp 19, the M absorption nozzle 20, the B lamp 21, the L absorption nozzle 22, and the R lamp 23 are sequentially installed on the ends of the six arms of the composite bracket 3, which can be connected by bolts. Tenon and tenon structure or magnetic adsorption can also be used. The 3-arm length of the composite bracket is 30-120mm, the width is 3-10mm, and the thickness is 0.3-1mm. The material is metal, preferably tool steel; the ring width in the structure shown in Figure 4 is 3-10mm, the thickness is 0.1-0.5mm, and the material can be Metal or organic materials, preferably tool steel; other parts are metal materials, preferably brass.

The microscope seat 4 is the bracket of the microscopic imaging mechanism and part of the lighting mechanism. In this embodiment, the shape is a rectangular parallelepiped, and the upper half is machined with inclined holes distributed on a conical surface for installing the lamp seat 24, the microscope 26 and the regulator 27. There is a hole for passing X-rays 25 at the position of the symmetry axis of each inclined hole, and the diameter of this hole is 3-10mm. Fig. 5 is a front view of this part. The microscope stand 4 is installed on the base plate 1 by bolts, and can also be directly installed on the working table of the X-ray diffractometer, with a height of 200-400mm, a width of 50-150mm, and a thickness of 50-100mm. Metal material, preferably duralumin.

The crystallization box 5 is used for growing high-quality protein crystals, and can be mounted on the gear shaft 16 and rotate in a vertical plane for inspection. The crystallization box 5 has a disc structure, and the crystallization ponds 31 are arranged in a concentric multi-circle array on it, as shown in FIG. X-ray 25 on the optical path. Crystal box 5 is 5-15mm in thickness, 100-200mm in diameter, 5-10mm in diameter for the central mounting hole, made of transparent polymer material, and injection-molded.

The large gear 6 is coaxial with the crystallizing box 5 for transmitting the rotation of the pinion 11 to the crystallizing box 5 through the toothed belt 7 . The large gear 6 is 15-30mm in diameter and 3-10mm in thickness, and is made of metal or polymer material, preferably brass.

The toothed belt 7 is used to transmit the rotation of the pinion 11 to the large gear 6, and is a standardized commercial product whose specifications are matched with the gears.

The base 8 is used for mounting and positioning the gear shaft 16, and the structure is shown in Figures 1 and 2, with a height of 80-150mm, a width of the mounting surface of 30-100mm, and a thickness of 5-10mm, and is made of metal, preferably duralumin.

The inspection motor 9 is used to provide power for the rotation of the crystallization chamber, preferably a stepping motor, with a rotation speed of 30-120 rpm and a step angle of 0.05-1 degrees.

Cantilever 10

The pinion gear 11 is used to transmit the rotational motion of the inspection motor 9 to the large gear 6 through the toothed belt 7 and then to the crystallizing box 5 . The pinion gear 11 is 8-20mm in diameter and 3-10mm in thickness, and is made of metal or polymer material, preferably brass.

The X slide rail 12 is used to realize the adjustment and positioning of the lateral position of the crystallizing box 5 in the plane parallel to the paper surface. Not less than 50mm.

The Y slide rail 13 is used to realize the adjustment and positioning of the longitudinal position of the crystallizing box 5 in the plane parallel to the paper surface. Not less than 100mm.

The X motor 14 is used to drive the X slide rail 12 to move, preferably a stepper motor with a rotational speed of 60-180 rpm and a step angle of 0.1-2 degrees.

The Y motor 15 is used to drive the Y slide rail 13 to move, preferably a stepper motor with a rotational speed of 60-180 rpm and a step angle of 0.1-2 degrees.

The gear shaft 16 is used to install the crystal box 5 and the large gear 6 on the base 8, the diameter is 4-10mm, and the metal material is preferably brass.

The lamp nozzle motor 17 is used to drive the composite bracket 3 to rotate to realize the replacement of the illuminating lamp and the absorbing nozzle.

The S-absorbing nozzle 18 is a small-diameter metal block that absorbs the X-rays 25 , and is cylindrical, and one end is machined with a conical pit. The S-absorbing nozzle 18 has a diameter of 0.1-0.5mm and a length of 5-15mm, and is made of lead alloy or tungsten alloy.

The G lamp 19 is an illuminating device of medium wavelength visible light used for transmitting illumination to the crystallization cell 31 , and usually emits green light, but can also be replaced with a white light lamp. LED lights are preferred, with a power of 0.05-0.5W.

The M absorption nozzle 20 is a metal block with a medium diameter for absorbing X-rays 25 , in the shape of a cylinder, and one end is machined with a conical pit. The M absorption nozzle 20 has a diameter of 0.5-1.0 mm and a length of 5-15 mm, and is made of lead alloy or tungsten alloy.

The B lamp 21 is an illuminating device for short-wavelength visible light that performs transmitted illumination on the crystallization cell 31 , and usually emits blue light, but can also be replaced with a white light lamp. LED lights are preferred, with a power of 0.05-0.5W.

The L-absorbing nozzle 22 is a large-diameter metal block that absorbs the X-rays 25 , and is cylindrical, and one end is machined with a conical pit. The L absorption nozzle 22 has a diameter of 1.0-1.5mm and a length of 5-15mm, and is made of lead alloy or tungsten alloy.

The R lamp 23 is an illuminating device of longer-wavelength visible light used for transmitting illumination to the crystallization cell 31 , and usually emits red light, but can also be replaced with a white light lamp. LED lights are preferred, with a power of 0.05-0.5W.

The lamp socket 24 is used for mounting the blue light 28, the green light 29 and the red light 30 and supplying power to them, and is cylindrical, 10-30mm in diameter, 50-100mm in length, and made of metal.

The X-ray 25 is not an essential part of the present invention, and is added for the convenience of describing the working principle of the device. It is not a solid part, but a beam of invisible light with a wavelength of 0.1-0.2nm and a beam diameter of 0.01-0.5mm. X-rays are harmful to the human body, and safety protection should be taken when people approach.

The microscope 26 is used to find and observe the protein crystals 34 in the crystallization tank 31, with a focal length of 100-300 mm, a physical magnification of up to 50 times, and a self-contained white light coaxial illumination, with a diameter of 10-30 mm.

Regulator 27

The blue light 28 emits blue light 35 after being powered on, and is used for illuminating the protein crystal 34 in an epi-radiation manner. Monochromatic LEDs are preferred, with a power of 0.05-0.5W.

The green light 29, which emits green light 35 after being powered on, is used to illuminate the protein crystal 34 in an epi-photographic manner. Monochromatic LEDs are preferred, with a power of 0.05-0.5W.

The red light 30 emits red light 35 after being powered on, and is used for illuminating the protein crystal 34 in an epi-radiation manner. Monochromatic LEDs are preferred, with a power of 0.05-0.5W.

Crystallization pool 31, a small pool for growing protein crystals 34 on the crystallization box 5, the opening is an isosceles trapezoid with a short centripetal side and a long opposite side, which contains protein solution 33 and balance solution 32, the structure is shown in Figure 7 and Figure 7 8, the interior has a stepped structure, the steps are on the outer peripheral side, the balance liquid 32 is in the space under the steps, the protein liquid 33 is in the pits on the steps, the number of pits is 1-3, and the pits are cylindrical or spherical. 1-3mm, opening diameter 2-4mm. The material is the same as that of the crystal box 5, and it is injection molded into one. The crystallization pools 31 are distributed in an equidistant array of concentric circles on the top of the crystallization box 5, the depth of the pool is 5-10mm, the length and width of the opening are 8-12mm, the height of the steps is 3-7mm, and the width of the steps is 3-5mm.

The balance solution 32 is an aqueous solution with a lower vapor pressure than the protein solution 33 , and is used to absorb the water vapor evaporated from the protein solution 33 .

The protein solution 33 is an aqueous solution in which high-purity active protein is dissolved, and usually also contains chemical reagents that buffer pH changes. The water in the protein solution 33 is continuously evaporated into vapor and absorbed by the equilibrium solution 32 to achieve supersaturation, and finally the protein crystal 34 is precipitated and gradually grows up.

The protein crystal 34 is a new phase in which the protein in the protein solution 33 reaches a supersaturated state due to the continuous evaporation of solvent water, and then precipitates in the form of a solid in which the molecules are arranged in an orderly manner.

The blue light 35 is not an essential part of the present invention, and is added for the convenience of describing the working principle of the device. It is not a solid part, but a beam of light used to illuminate the protein crystal 34 .

The green light 36 is not an essential part of the present invention, and is added for the convenience of describing the working principle of the device. It is not a solid part, but a beam of light for illuminating the protein crystal 34 .

The red light 37 is not an essential part of the present invention, and is added for the convenience of describing the working principle of the device. It is not a solid part, but a beam of light used to illuminate the protein crystal 34 .

The white light 38 is not an essential part of the present invention, and is added for convenience of describing the working principle of the device. It is not a solid part, but a beam of light used to illuminate the protein crystal 34 .

The above-mentioned embodiments only represent the preferred embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as limiting the scope of the patent of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications, improvements and substitutions can be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (10)

1. A device for in-situ X-ray diffraction of protein crystals, comprising an X slide rail (12) driven by an X motor (14), a Y slide rail (13) driven by a Y motor (15), and a Y slide rail (13) driven by an X motor (14), The inspection motor (9) on the cantilever (10) drives the inspection and positioning mechanism through a pinion (11), a toothed belt (7) and a large gear (6) mounted on the gear shaft (16), and a base plate (1) a support mechanism composed of a base (2), a microscope base (4) and a base (8), characterized in that the device further comprises a disc-shaped crystallizing box (5), a crystallizing tank (31), a balance Crystallization chamber mechanism composed of liquid (32), protein liquid (33) and protein crystal (34), and a microscopic imaging mechanism composed of a microscope (26) for observing and identifying crystals and its regulator (27), which is composed of a G lamp (19), B lamp (21), R lamp (23), lamp holder (24), blue light (28), green light (29), red light (30) The lighting mechanism is composed of a composite bracket ( 3) The lamp nozzle motor (17), the S absorption nozzle (18), the M absorption nozzle (20), the L absorption nozzle (22) and the X-ray (25) constitute a diffraction absorption mechanism. 2. The device for in-situ X-ray diffraction of protein crystals according to claim 1, wherein the blue light (35) emitted by the blue light (28), the green light (36) emitted by the green light (29), The red light (37) emitted by the red light lamp (30) and the white light (38) emitted by the microscope (26) are in a focused distribution, and together with the X-rays (25), they all irradiate the same protein solution (33) and protein crystals ( 34) on. 3. The device for in-situ X-ray diffraction of protein crystals according to claim 1, wherein the disc-shaped crystallization box (5) is mounted on the gear shaft (16) through the middle mounting hole, and can be inspected by The motor (9) is driven to rotate in a vertical plane by the power transmitted from the pinion gear (11), the toothed belt (7), the large gear (6) and the gear shaft (16). 4. The device for in-situ X-ray diffraction of protein crystals according to claim 1, wherein the illumination mechanism is simultaneously equipped with epi- and transmission multi-color illumination, G lamp (19), B lamp (21) and R lamp ( 23) is a transmitted illumination light source, the blue light (35) emitted by the blue light (28), the green light (36) emitted by the green light (29), the red light (37) emitted by the red light (30), and the microscope (26) ) emitted white light (38) is an epi-illumination light source. 5. The device for in-situ X-ray diffraction of protein crystals according to claim 1, wherein the ends of the arms of the composite support (3) are simultaneously equipped with G lamps (19), B lamps (21) and The R lamp (23), and the S absorption nozzle (18), the M absorption nozzle (20) and the L absorption nozzle (22) for absorbing X-rays (25) are driven by the lamp nozzle motor (17) to rotate the composite bracket (3) , to realize the replacement of lighting lamps and suction nozzles. 6. The device for in-situ X-ray diffraction of protein crystals according to claim 3, wherein the crystallization box (5) is disc-shaped, and the crystallization pools (31) on it are arranged in a concentric circle array, and the protein The liquid (33) is located on the inner and outer steps of the crystallization tank (31); the thickness of the crystallization box (5) is 5-15mm, the diameter is 100-200mm, and the diameter of the central mounting hole is 5-10mm; And the isosceles trapezoid with the length of the opposite side, the depth is 5-10mm, the length and width of the opening are 8-12mm, the height of the step is 3-7mm, and the width of the step is 3-5mm. 7. The device for in-situ X-ray diffraction of protein crystals according to claim 5, wherein the six arms of the composite support (3) are radially distributed at equal included angles, and a ring is used to connect the ends of each arm. In one piece, the arm length is 30-120mm, the width is 3-10mm, the thickness is 0.3-1mm, and the material is metal. 8. The device for in-situ X-ray diffraction of protein crystals according to claim 5, characterized in that the number of arms of the composite support (3) is greater than six, and is radially distributed at equal included angles, and a circular ring is used to separate the arms of each The end of the arm is connected into one body, the arm length is 30-120mm, the width is 3-10mm, the thickness is 0.3-1mm, and the material is metal. 9. The device for in-situ X-ray diffraction of protein crystals according to claim 5, characterized in that the number of arms of the composite support (3) is less than six, and is radially distributed at an equal angle, and a ring is used to separate the arms of each The end of the arm is connected into one body, the arm length is 30-120mm, the width is 3-10mm, the thickness is 0.3-1mm, and the material is metal. 10. The device for in-situ X-ray diffraction of protein crystals according to any one of claims 7-9, wherein the material of the arms of the composite support (3) is tool steel.

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