CN110256527B - Protein composite crystal growing box for in-situ X-ray diffraction - Google Patents

Abstract

The invention discloses a protein composite crystal growing box for in-situ X-ray diffraction, which aims to solve the technical problem that protein crystals are required to be fished out from a crystallization chamber to implement X-ray diffraction in the technical field of biology and possibly damage the crystals, and provides a more effective means for researching the structure and function of protein molecules and further developing intelligent medicaments. The composite crystal cultivating box consists of a plurality of composite crystallization chambers and/or a plurality of vapor phase crystallization chambers which are arranged in an array and magnetic materials, wherein porous materials for adsorbing and fixing precipitation liquid or equilibrium liquid, gel thin layers for adsorbing and fixing protein liquid and protein crystal and baffles for isolating the protein liquid from the precipitation liquid or the equilibrium liquid are arranged in the two crystallization chambers; the baffle has notches or gaps to make the protein liquid contact with the precipitating liquid directly or via vapor phase. As a result of the contact, the protein solution becomes supersaturated and protein crystals precipitate. When in use, the crystal is not needed to be fished out, and the crystal cultivating box is directly placed on the X-ray diffractometer in a side-standing manner.

Description

Protein composite crystal growing box for in-situ X-ray diffraction

Technical Field

The invention discloses a composite crystal growing box for directly placing protein crystals growing in a solution and a container in X-ray to implement in-situ diffraction, belonging to the technical field of biology.

Background

The life sciences are the science of the twenty-first century, and the life science revolution triggered by the human genome project reaches the climax in the structural genome or proteome period of the post-genome era, because genes are the blueprints of life, and proteins are the realization of life. Proteins are both "bricks" that construct living bodies and "molecular machines" that perform vital activities.

The function of the molecular machinery of a protein depends on its molecular structure. Protein molecules are very complex, typically consisting of thousands of atoms of carbon, hydrogen, oxygen, nitrogen, sulfur, etc. The molecular structure of proteins can be classified into class I, class II, class III, and class IV structures. The I-stage structure is in a non-branched linear shape, and is formed by connecting carboxyl and hydroxyl of the same or different amino acids through a condensation reaction to generate a peptide bond, which is called a peptide chain. The II-stage structure is a typical pattern formed by folding the I-stage structure, such as a spiral shape and a sheet shape. The III-level structure of the protein molecule is a three-dimensional space structure formed by an ordered II-level structure and a disordered part of a peptide chain, and the protein molecule has specific functions. The class IV structure refers to a complex formed by assembling several peptide chains each having its own class III structure, and a single peptide chain is called a subunit. It has been found that the class III structure of protein molecules is of great importance, and different class I structures may have similar class III structures so that they function similarly, while different class III structures determine different biological functions. Therefore, the method for revealing and researching the III-level structure of the protein molecule has important significance for further researching the biological function, the pathological mechanism and the development of intelligent protein drugs.

Research to determine the class III structure of protein molecules is the core work of structural genomics or proteomics. Currently there are mainly four methods: crystal X-ray diffraction method, NMR nuclear magnetic resonance method, freezing transmission electron microscopy, and theoretical prediction method. The crystal X-ray diffraction method is the most important and commonly used method at present, and is to assemble protein molecules into a single crystal, the single crystal is irradiated by X-rays to obtain a diffraction pattern containing molecular structure information, and the diffraction pattern is subjected to a series of crystallography calculation analysis, so that the 'Lushan Zhenliang' of the three-dimensional space structure of the protein molecules is finally reduced. Due to a series of reasons of many protein molecule atoms, loose structure, high water content and the like, the growth of large-size and high-quality crystals is very difficult. Researchers have developed various methods such as vapor phase diffusion crystallization, liquid preparation crystallization, dialysis crystallization, etc., and even have carried out crystallization on the ground, in order to improve the growth size and quality of protein crystals by means of microgravity to inhibit convection in protein liquid. Among these methods, the vapor phase crystallization method and the liquid phase crystallization method are most commonly used, and different methods are sometimes selected for different proteins.

With the improvement of the power and the quality of an X-ray light source, like the use of a synchronous radiation light source, the requirement on the size of a protein crystal is reduced from a millimeter level to a micron level, but the following problems are that the operations of crystal fishing, fixing, liquid nitrogen freezing, centering adjustment and the like are more difficult.

Therefore, the main manufacturer for producing desktop-level X-ray light sources, Rigaku corporation, Japan, proposes a technology of directly placing a high-flux crystallization plate in an X-ray path to perform diffraction, and develops a crystallization plate clamping and positioning mechanism, and does not use a nylon wire ring with a micron diameter to fish and freeze crystals. But no special high-flux crystallization plate which can be placed on one side is available in the market at present; the invented in-situ diffraction crystallization box is described in the published invention patent of 'an in-situ diffraction device and diffraction method of protein crystal' (publication No. CN108593689A) and 'a diffraction method of biological macromolecular crystal in a near physiological state' (publication No. CN108732193A), but the technical scheme of sticking high molecular films on two sides of a double-sided adhesive tape can not fix the crystal when the box is used on one side, and the double-sided adhesive tape has the problem of physicochemical compatibility with a plurality of protein crystallization solutions, and in addition, the technical scheme does not give the layout scheme of the protein solution and a balance liquid/precipitation liquid thereof, namely, does not clearly adopt which crystallization method, such as a vapor phase diffusion method, a liquid-liquid diffusion method or a liquid preparation method and the like. When a common crystallization plate is placed on one side, protein crystals move, equilibrium liquid and protein liquid move, X-rays are diffracted by the crystals and then change directions and are blocked by the side wall of the crystallization chamber, the quality of diffraction patterns is reduced by light people, and wrong information is introduced to cause incorrect structure of resolved protein molecules when serious people suffer from the problems. Therefore, the development of an in situ diffracting crystallization tool becomes a key to solving the current problems.

Disclosure of Invention

In order to solve the problems and the defects, the invention discloses a protein composite crystal cultivating box which integrates two protein crystallization methods of vapor diffusion and liquid phase diffusion and is suitable for different protein requirements; meanwhile, the crystal cultivating box reduces the bottom plates for supporting the protein liquid in the composite crystallization chamber and the vapor phase crystallization chamber so as to eliminate the blockage of the side walls to the diffracted X-rays, and adds a flexible hydrophilic layer between the protein liquid and the chamber bottom for adsorbing and fixing protein crystals; moreover, in order to prevent the sediment solution or the balance solution from flowing and interfering the protein solution when the crystal cultivating box is erected on one side, the crystal cultivating box is also provided with 1 liquid absorption or solid-liquid material; in addition, the magnetic material is embedded in the corner groove of the crystal growing box, so that the crystal growing box is convenient to assemble and disassemble on the in-situ diffraction clamp.

The invention is suitable for the crystal cultivation of protein crystal in-situ X-ray diffraction, and can overcome the problems of blocking X-ray, movement of precipitation liquid or equilibrium liquid, movement of crystal and the like. The new crystal-cultivating box is also suitable for the cultivation of protein crystals without in-situ X-ray diffraction, the new crystal-cultivating box can prevent the displacement of the precipitation liquid or the equilibrium liquid which is possibly caused by improper moving operation, and the flexible hydrophilic layer is convenient for fishing the protein crystals by using micron-diameter nylon wire rings without damaging the crystals. At present, similar products do not exist at home and abroad, the complex operations such as fishing of tiny protein crystals, freezing of liquid nitrogen, transferring, storage of liquid nitrogen and the like and the risk of possible crystal damage are avoided when new products are put into operation, and the development and use of the new crystal cultivating box have remarkable economic and social benefits in consideration of the capital cost, time cost and labor cost of the preparation of the early-stage protein and reagent consumables.

The innovation points of the invention are as follows:

(1) fixing the protein crystal by means of the hydrophilic net material between the protein liquid and the bottom of the crystallization chamber; (2) fixing the protein liquid by means of a hydrophilic net-shaped supermolecular structural material or a three-dimensional net between the protein liquid and the bottom of the crystallization chamber; (3) adopting an open porous material or a hydrophilic net-shaped supermolecular structure material to fix the precipitation solution or the equilibrium solution; (4) the height of the part of the pool bottom for supporting the protein liquid is reduced so as to eliminate the blockage of the side wall of the crystallization chamber to the diffraction X-ray; (5) the crystal cultivating box is fixed on the clamping mechanism by using a magnetic material, so that the crystal cultivating box is convenient to assemble and disassemble; (6) the method has two crystallization methods of vapor phase crystallization and composite crystallization, and particularly, the composite crystallization organically integrates the traditional vapor phase crystallization and the traditional liquid phase crystallization together, so that the advantages are complemented.

Drawings

FIG. 1 is a front perspective view of a cassette according to the present invention;

FIG. 2 is a partial structure diagram of the section A-A in FIG. 1;

FIG. 3 is a schematic perspective view of a composite crystallization chamber;

FIG. 4 is a schematic perspective view of a vapor phase crystallization chamber;

FIG. 5 is a schematic view of a vapor phase crystallization chamber showing solvent diffusion;

FIG. 6 is a schematic view of solvent diffusion in a composite crystallization chamber;

FIG. 7 is a schematic view of a vapor phase crystallization chamber of a conventional structure blocking X-rays;

FIG. 8 is a schematic diagram of the novel vapor phase crystallization chamber with X-ray blockage removed.

Detailed Description

In order that those skilled in the art can better understand the present invention, the following technical solutions are further described with reference to the accompanying drawings and examples.

1. Composition of

1) The overall structure of the crystal growing box is as follows:

fig. 1 and 2 show a preferred embodiment of the crystal growing box, briefly, the crystal growing box is composed of a box body, a composite crystallization chamber, a vapor phase crystallization chamber and a magnetic material, adopts a bilateral symmetry structure layout, and is composed of a box body 1, a composite crystallization chamber 2, a vapor phase crystallization chamber 3 and a magnetic material 4; FIG. 2 is a schematic view of a portion of the cross section of the incubator A-A near the edge. In the scheme, the box body 1, the baffle 7, the U-shaped plate 13, the light-transmitting bottom plate 17 and the liquid bearing plate 18 are of an integral structure and are formed in one step through an injection molding process. Another alternative may also be taken: the composite crystallization chamber 2 and the vapor phase crystallization chamber 3 are respectively small containers which are completely independent, and the box body 1 is only a bracket for fixing the composite crystallization chamber 2 and the vapor phase crystallization chamber 3, but the structural performance, the cost advantage and the like of the scheme are not the same as those of the above preferred scheme, so the details are not repeated.

2) The structure and the working principle of the composite crystallization chamber 2 are as follows:

the structure is as follows: the composite crystallization chamber 2 is composed of a solid-liquid material 5, a precipitation liquid 6, a baffle 7, a liquid-phase protein liquid 8, a solid crystal layer 9, a liquid-phase protein crystal 10 and a liquid bearing plate 18. FIG. 3 is a schematic perspective view of the complex crystallization chamber 2 in which the right side and the upper side of the baffle 7 are not closed; the width of the liquid phase protein liquid 8 can be the same at the left end and the right end, and can also be the left end and the right end, so as to adjust the mild degree of liquid-liquid diffusion between the liquid phase protein liquid 8 and the precipitation liquid 6. Alternatively, the right side and the upper side of the baffle 7 can be closed, and a channel for water molecule diffusion and transfer is formed by opening some regular or irregular holes on the baffle, so that the baffle becomes simple liquid phase diffusion or has a composite function of vapor phase diffusion.

The working principle is as follows: the right side and the upper side of the baffle 7 in the schematic perspective view of the composite crystallization chamber 2 shown in FIG. 3 are not closed, and the liquid-phase protein solution 8 is in direct contact with the precipitation solution 6 in the solid-liquid material 5 through a gap on the right side and in indirect contact through a vapor phase through a gap on the upper side. As shown in fig. 6, the design enables the water in the liquid-phase protein liquid 8 to be transferred to the precipitation liquid 6 through the contact surface with the precipitation liquid 6 in a liquid-liquid diffusion manner, so as to realize the concentration and supersaturation of the liquid-phase protein liquid 8, and further separate out the liquid-phase protein crystal 10; the water in the liquid-phase protein liquid 8 can be evaporated into gas, and is absorbed by the precipitation liquid 6 through the upper side gap, so that the concentration and supersaturation of the liquid-phase protein liquid 8 are realized, and finally, the liquid-phase protein crystal 10 is separated out. In order to prevent the liquid-phase protein crystal 10 from moving when the crystal cultivating box 1 is used in a side-standing mode, a layer of hydrophilic material, namely a crystal fixing layer 9 is arranged on the inner surface of a liquid bearing plate 18 of the composite crystallization chamber 2, and the crystal grows gradually on the surface and is slowly embedded into the crystal fixing layer to realize the fixation of the crystal; the fixed liquid phase protein liquid 8 can adopt gel, and can not be fixed when the liquid is little; the solid-liquid material 5 is fixed to the precipitation liquid 6.

3) The structure and the working principle of the vapor phase crystallization chamber 3 are as follows:

the structure is as follows: the vapor phase crystallization chamber 3 is composed of a liquid absorbing material 11, a balance liquid 12, a U-shaped plate 13, a liquid absorbing layer 14, vapor phase protein liquid 15, vapor phase protein crystal 16 and a light-transmitting bottom plate 17. FIG. 4 is a schematic perspective view of the vapor phase crystallization chamber 3, wherein a U-shaped plate 13 is provided with a channel for water molecules to evaporate from the vapor phase protein liquid 15 and to be absorbed by the equilibrium liquid 12; a plurality of drops of vapor phase protein liquid 15 can be spotted on the inner surface of the light-transmitting bottom plate 17; and (3) mounting a crystal absorption layer 14 at the position where 15 drops of the vapor phase protein liquid are supposed to be discharged on the light-transmitting bottom plate 17.

The working principle is as follows: fig. 5 is a schematic diagram of the diffusion of solvent in the vapor phase crystallization chamber, which can show the working principle more clearly, the solvent in the vapor phase protein liquid 15 is evaporated into vapor, and then absorbed by the exposed equilibrium liquid 12 through the gap on the U-shaped plate 13, and the continuous process will concentrate the vapor phase protein liquid 15 continuously, make it supersaturated, and finally precipitate the vapor phase protein crystal 16 in the interior. In order to avoid the displacement of the vapor phase protein crystal 16 when the crystal cultivating box 1 is laterally diffracted, a layer of hydrophilic material, namely a crystal absorption layer 14, is coated on the inner surface of a light-transmitting bottom plate 17 of the vapor phase crystallization chamber 3, and the crystal gradually grows on the surface and slowly sinks in to realize the positioning of the crystal; the fixed vapor phase protein liquid 15 can adopt gel or thickening agent, and no measures can be taken when the liquid is little; holding the balancing liquid 12 is a liquid absorbent material 11.

2. Structure of each part

The crystal growing box body 1 can be made of metal or organic materials, preferably thermoplastic transparent organic materials, and the softening temperature of the materials is not lower than 70 ℃. In view of the matching use with the existing desktop-level X-ray diffractometer, the overall size thereof should be controlled in the following range: the length is less than 200mm, the width is less than 150mm, and the thickness is less than 20 mm. The structure of the crystal growing box body 1 can adopt an integral injection molding structure as described above, the box body 1, the baffle 7, the U-shaped plate 13, the light-transmitting bottom plate 17 and the liquid bearing plate 18 are integrated, and the injection molding surface roughness is superior to 6.3 mu m. The second option can be a split module combination, the baffle 7, the U-shaped plate 13, the light-transmitting bottom plate 17 and the liquid-bearing plate 18 are not integrated with the box body 1, wherein the baffle 7 and the liquid-bearing plate 18 are integrated in the independent module of the composite crystallization chamber 2, the U-shaped plate 13 and the light-transmitting bottom plate 17 are integrated in the independent module of the vapor phase crystallization chamber 3, and the box body 1 is a frame structure with a square hole array.

In the integrated structure, the liquid bearing plate 18 of the composite crystallization chamber 2 and the light-transmitting bottom plate 17 of the vapor phase crystallization chamber 3 are changed from the traditional half-height position to be flush with the bottom surface of the crystal growth box body 1, as shown in fig. 7 and 8, the traditional structure can shield diffraction X-rays such as R2 and R3, and the new design structure can effectively avoid shielding, so that diffraction lines R2 and R3 containing protein crystal structure information are effectively preserved.

The composite crystallization chamber 2 is one of the core of the device, when the crystal growing box body 1 is of an integrated structure, the composite crystallization chamber 2 is not a solid component which coexists with other components, and is composed of a part of the crystal growing box body 1, a baffle 7 and a liquid bearing plate 18, and the structure is as shown in fig. 2, fig. 3 and fig. 6. The length, width and depth/height of the composite crystallization chamber 2 are controlled within 15mm, and the width of the gap on the upper edge and the side surface of the baffle 7 is 1-5 mm. For a modular cassette configuration, the composite crystallization chamber 2 will be a separate module, as previously described.

The vapor phase crystallization chamber 3 is another core component of the device, when the crystal growing box body 1 adopts a preferable integrated structure, the vapor phase crystallization chamber 3 is not a solid component like other components, and is formed by a part of the crystal growing box body 1, a U-shaped plate 13 and a light-transmitting bottom plate 17, and the structure is shown in fig. 2, fig. 4 and fig. 5. The length, width and depth/height of the vapor-phase crystallization chamber 3 are generally controlled within 15mm, and the U-shaped plate 13 divides the inner space of the vapor-phase crystallization chamber 3 into two parts. For the modular cassette configuration, the vapor phase crystallization chamber 3 will also become a separate module, as previously described.

The magnetic material 4 is a cylinder or a cuboid, preferably a neodymium iron boron permanent magnet material, the diameter of the cylinder is 3-5mm, the length of the cylinder is 5-10mm, the length, the width and the height of the cuboid are respectively 5-15mm, 3-5mm and 5-10mm, and the surface of the cuboid is wrapped with a plastic film with the thickness of less than 1 mm. The magnetic material 4 is clamped in a groove on the inner side of the corner of the box body 1 of the crystal growing box in an interference fit manner, and can also be bonded and fixed through an adhesive.

The solid-liquid material 5 is used for fixing the precipitation liquid 6, is a high-elasticity porous material, needs to have good compatibility with the precipitation liquid 6, and has internal pores with open pores communicated with each other, and the cross section diameter of each pore is not more than 0.5 mm. The shape and size of the solid-liquid material 5 can be cut at any time from the bulk material according to the actual internal space requirement of the composite crystallization chamber 2. Organic high molecular material, preferably silicon rubber material, can be selected.

The precipitation liquid 6 is a liquid with vapor pressure difference or component concentration difference with the liquid-phase protein liquid 8, and the two have matching property. The precipitation liquid 6 is not an essential structural component of the invention, but is here given as a component part for the sake of convenience in illustrating the principle and the method of use. The precipitation liquid 6 functions to absorb water vapor evaporated from the liquid-phase protein liquid 8 and/or wherein high concentrations of components enter the liquid-liquid diffusion occurring upon contact with the liquid-phase protein liquid 8, with the same effect of gradually supersaturating the liquid-phase protein liquid 8 to precipitate liquid-phase protein crystals 10.

A baffle 7: the function is to separate the precipitation liquid 6 from the liquid phase protein liquid 8, and only a gap is left on one side surface and the upper edge, so that the two liquids are directly contacted at the side surface and indirectly communicated with each other at the top by virtue of a vapor phase, namely two channels of liquid-liquid diffusion and vapor diffusion are established, as shown in figure 6. The preferable scheme is that the baffle 7 and the crystal cultivating box body 1 are integrally injected, the bottom and one side surface are connected together, the gap at the other side is 1-5mm, and the gap at the upper edge is 1-5 mm. The baffle 7 may be parallel to the side wall of the composite crystallization chamber 2 or not, for example, a structure with a narrower end having a slit on the side surface is adopted to realize a specific liquid-liquid diffusion effect, which is beneficial to the growth and the internal quality of the liquid-phase protein crystal 10. The baffle 7 preferably has a thickness in the range of 0.1-0.5 mm.

The liquid phase protein solution 8, like the precipitation solution 6, is not an essential structural component of the present invention, but is provided as a component for convenience of illustration of the principle and method of use. The concentration and the volume of the liquid phase protein liquid 8 are determined according to the actual experiment requirements.

A solid crystal layer 9: the function is to fix the liquid-phase protein crystal 10 separated out from the liquid-phase protein liquid 8, avoid the moving position, the thickness of the layer is 0.2-1mm, organic or inorganic material with three-dimensional net-shaped molecular structure is adopted, preferably agarose gel or polyacrylamide gel, and the mass concentration of the gel is 0.5-1.5%.

The liquid phase protein crystal 10 is protein crystal precipitated by orderly aggregation of protein molecules in the liquid phase protein liquid 8, is not an essential component of the invention, and is added for conveniently describing the working principle.

The liquid absorbing material 11 is used for adsorbing and fixing the equilibrium liquid 12, is an open porous organic or inorganic material, does not react with the equilibrium liquid 12, has a porosity of more than 90 percent and a pore diameter of less than 1 millimeter, and is preferably polyethylene or polycarbonate.

The balancing liquid 12, which is a liquid having a smaller vapor pressure difference than the vapor-phase protein liquid 15, is used to absorb water evaporated from the vapor-phase protein liquid 15, and the balancing liquid 12 is not an essential component of the present invention and is added for the convenience of explaining the working principle.

The U-shaped plate 13 serves to separate the balancing liquid 12 from the vapor-phase protein liquid 15, leaving gaps only in the middle or corner positions, so that water vapor evaporated from the vapor-phase protein liquid 15 can reach the balancing liquid 12 through the gaps and be absorbed by the balancing liquid, i.e., forming vapor diffusion channels, as shown in fig. 5. The preferred scheme is that the U-shaped plate 13 and the crystal cultivating box body 1 are integrally injected, the bottom and the side surfaces are connected together, a rectangular gap is arranged from the upper edge to the center, the length is 5-8mm, and the width is 3-6 mm. The U-shaped plate 13 is generally parallel to the side wall of the vapor-phase crystallization chamber 3 and may not be parallel. The U-shaped plate 13 preferably has a thickness of 0.1 to 0.5 mm.

The seed crystal layer 14 serves to anchor the vapor phase protein crystals 16 precipitated from the vapor phase protein liquid 15 so as not to change their positions. The seed layer 14 is preferably 0.2 to 1mm thick, and may be larger or smaller. The material is organic or inorganic material with a three-dimensional net-shaped microstructure, agarose gel or polyacrylamide gel is preferred, and the mass concentration of the gel is 0.3-1.2%.

The vapor phase protein fluid 15, which is not an essential structural component of the present invention, is provided as a component herein for purposes of clarity in illustrating the principles and methods of use of the present invention. The composition, concentration, volume and other parameters of the vapor phase protein liquid 15 are determined according to the actual experiment requirements.

The vapor phase protein crystal 16 is protein crystal formed by orderly arranging protein molecules in the vapor phase protein liquid 15, is not an essential component of the invention, and is added for convenience of describing the working principle.

The light-transmitting bottom plate 17 is the bottom of the vapor phase crystallization chamber 3 and is not an independent part when the composite crystal growing box 1 adopts an integrated injection molding structure. The light-transmitting bottom plate 17 is made of the same material as the composite crystal growing box 1, and when the vapor phase crystallization chamber 3 is an independent module, the material can be made of different materials, such as quartz glass. The thickness of the plate is 0.2-1.0 mm.

The liquid bearing plate 18 is the bottom of the composite crystallization chamber 2 and is not an independent structural component when the composite crystallization box 1 is in an integrated injection molding structure. The liquid bearing plate 18 is made of the same material as the composite crystal growing box 1, and when the composite crystallization chamber 2 is an independent module, different materials, such as quartz glass, can be adopted. The thickness of the plate is 0.2-1.0 mm.

The above examples are merely representative of preferred embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the present invention. It should be noted that, for those skilled in the art, various changes, modifications and substitutions can be made without departing from the spirit of the present invention, and these are all within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (7)

1. A protein composite crystal growing box for in-situ X-ray diffraction comprises a composite crystal growing box body (1) consisting of a composite crystallization chamber (2) array, a vapor phase crystallization chamber (3) array and a magnetic material (4), and is characterized by further comprising a solid-liquid material (5), a precipitation liquid (6), a baffle plate (7), a liquid phase protein liquid (8), a crystal fixing layer (9), a liquid phase protein crystal (10) and a liquid bearing plate (18) which form the composite crystallization chamber (2), a liquid absorbing material (11), a balance liquid (12), a U-shaped plate (13), a crystal absorbing layer (14), a vapor phase protein liquid (15), a vapor phase protein crystal (16) and a light-transmitting bottom plate (17) which form the vapor phase crystallization chamber (3), wherein the baffle plate (7) in the composite crystallization chamber (2) separates the precipitation liquid (6) from the liquid phase protein liquid (8), but gaps are left on the upper side and one side of the baffle plate (7), so that the precipitation liquid (6) in the solid-liquid material (5) can be directly contacted with the liquid-phase protein liquid (8) to realize liquid-liquid diffusion and can also realize vapor diffusion by means of vapor phase communication, composite crystallization simultaneously carried out by the two methods is realized, the solid crystal layer (9) is arranged on the inner surface of the liquid bearing plate (18), the U-shaped plate (13) in the vapor phase crystallization chamber (3) isolates the balance liquid (12) from the vapor-phase protein liquid (15), a notch or a hole on the U-shaped plate (13) can ensure that the balance liquid (12) in the liquid absorbing material (11) is communicated with the vapor-phase protein liquid (15) by means of vapor phase to realize vapor diffusion crystallization, the position of a quasi-point vapor-phase protein liquid (15) drop on the light-transmitting bottom plate (17) is provided with the liquid absorbing layer (14), the magnetic material (4) is fixed in a liquid absorbing corner groove of the composite crystallization box body (1), the solid-liquid material (5) fixes the precipitation liquid (6) and the balance liquid (12) is Does not flow and mix with the liquid phase protein liquid (8) or the vapor phase protein liquid (15) to destroy the crystal.

2. The protein composite crystallization cassette according to claim 1, wherein the baffle (7) in the composite crystallization chamber (2) is integrally molded with the cassette body (1) of the composite crystallization cassette, the bottom and one side are connected together, and the thickness of the baffle (7) is in the range of 0.1-0.5 mm.

3. The protein composite crystallization cassette according to claim 1, wherein a U-shaped plate (13) in the vapor phase crystallization chamber (3) is integrally injection-molded with the cassette body (1) of the composite crystallization cassette, the bottom and the side are connected together, and the thickness of the U-shaped plate (13) is 0.1-0.5 mm.

4. The protein composite crystal-growing box according to claim 1, characterized in that the composite crystal-growing box body (1) can be placed on the side to perform in-situ diffraction on an X-ray diffractometer, and the liquid-phase protein crystal (10) and the vapor-phase protein crystal (16) are adsorbed and fixed by the crystal-fixing layer (9) and the crystal-adsorbing layer (14) so that the crystals are still and convenient to diffract when the composite crystal-growing box body (1) is placed on the side.

5. The protein composite crystal growth box according to claim 1, characterized in that the magnetic material (4) can fix the composite crystal growth box body (1) on a clamp without tools.

6. The protein composite crystal growth box according to claim 2, characterized in that the liquid bearing plate (18) is positioned flush with the bottom surface of the composite crystal growth box body (1) to ensure that the diffraction lines generated by the X-rays after passing through the liquid-phase protein crystal (10) are not blocked by the side wall of the composite crystallization chamber (2).

7. The protein composite crystal growth box according to claim 3, characterized in that the light-transmitting bottom plate (17) is positioned flush with the bottom surface of the composite crystal growth box body (1) to ensure that the diffraction lines generated by the X-rays after passing through the vapor-phase protein crystals (10) are not blocked by the side wall of the vapor-phase crystallization chamber (2).

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