CN110361407B - Device for protein crystal in-situ X-ray diffraction - Google Patents
Device for protein crystal in-situ X-ray diffraction Download PDFInfo
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- CN110361407B CN110361407B CN201910666162.1A CN201910666162A CN110361407B CN 110361407 B CN110361407 B CN 110361407B CN 201910666162 A CN201910666162 A CN 201910666162A CN 110361407 B CN110361407 B CN 110361407B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
- G01N23/20008—Constructional details of analysers, e.g. characterised by X-ray source, detector or optical system; Accessories therefor; Preparing specimens therefor
- G01N23/20025—Sample holders or supports therefor
Abstract
The invention discloses a device for protein crystal in-situ X-ray diffraction, which is used for solving the problems of difficulty in fishing, identifying and positioning diffraction of a tiny protein crystal and the like in the field of biotechnology and improving the utilization rate of the crystal. The device consists of a two-dimensional translation and one-dimensional rotation inspection positioning mechanism, a disc-shaped crystallization box mechanism, an illuminating mechanism, a micro-imaging mechanism, a diffraction absorption mechanism and a supporting mechanism. The inspection positioning mechanism carries out two-dimensional translation positioning on the disc-shaped crystallization box, the inspection motor drives the crystallization box to rotate so as to inspect the crystallization tank array distributed on the crystallization box in a concentric circle, and the crystallization box does not need to be turned over in the middle; finding out protein crystals by using the falling rays or the transmission illumination of the illumination mechanism and the lamplight with different colors through a long-focus microscope, and enabling the X-rays to accurately pass through the crystals by means of fine adjustment of the routing inspection positioning mechanism; the X-rays traveling straight through the crystal are absorbed by the absorption nozzles, wherein the absorption nozzles of different specifications can be automatically selected and replaced according to the size of the protein crystal.
Description
Technical Field
The invention discloses a device capable of installing protein crystals and a crystallization box on an X-ray diffractometer to implement in-situ diffraction, belonging to the technical field of biology.
Background
The life science is the most challenging science at present, genome and proteome research provides infinite possibilities for developing almost inexhaustible biotechnological products, and the dream of longevity of human beings, early diagnosis and treatment of serious diseases, killing or modified domestication of bacteria and viruses are no longer the illusion of delusions. The development of these biotechnology requires fine atomic structural information on protein molecular machines, because proteins are not only structural substances of organisms, but also executives of life activities, and are a delicate molecular machine.
The molecular machinery of proteins is very complex, consisting of thousands or more of atoms of carbon, hydrogen, oxygen, nitrogen, etc. There are four main methods for determining the atomic-scale structure of the molecular machinery of proteins: crystal X-ray diffraction, nuclear magnetic resonance, low-temperature transmission electron microscope and theoretical calculation. The crystal X-ray diffraction method is the most common and effective method at present, but the precondition is to crystallize protein and obtain high-quality single crystals, and the cultivation of millimeter-scale high-quality single crystals is particularly difficult due to the reasons of many atoms contained in protein molecules, large structural flexibility, large amount of bound water and free water, and the like. For this reason, various methods such as vapor diffusion crystallization, liquid-liquid diffusion crystallization, liquid preparation method crystallization, dialysis crystallization and the like have been developed, and even proteins are sent to the outer space in order to improve the growth size and quality of protein crystals by means of the inhibition of convection in protein liquid by microgravity. Among these methods, the vapor phase crystallization method and the liquid-liquid crystallization method are most commonly used, and different methods are sometimes selected for different proteins.
With the development of science and technology, the power and quality of X-rays are greatly improved, for example, the use of synchrotron radiation and free electron laser light sources reduces the requirements on protein crystals from millimeter level to micron level, but the operations of fishing, soaking, liquid nitrogen freezing and the like of micron level crystals are easy to damage the crystals, so that the previous work is abandoned.
Therefore, the main manufacturer for manufacturing laboratory-level X-ray diffraction equipment, Rigaku corporation, Japan, proposed a technique of directly mounting a multi-well crystallization plate on an X-ray diffractometer to perform diffraction, and developed a crystallization plate fixing and adjusting mechanism, which can fish out crystals without using a micron-sized nylon wire ring and perform soaking and liquid nitrogen freezing. But the technologies only have the falling radiation on the micron-sized crystal, the effect is poor, and the crystal is difficult to find and position; the X-ray absorption nozzles with different specifications are used according to the sizes of the crystals and need to be replaced manually, and the X-rays need to be closed; at present, no special crystallization plate which can be placed on one side in mass production is available in the market. The key of the technical scheme described in the published invention patent of 'in-situ diffraction device and diffraction method of protein crystal' (publication number CN108593689A) is a crystallization box for in-situ diffraction, and no technical description about the fixation of the crystallization box and the observation of the crystal and the matching of the device and an X-ray diffractometer is provided; according to the technical scheme, the polymer films are adhered to two sides of the double-sided adhesive tape used by the crystallization box, when the box is used on the side, the crystal cannot be fixed, the micro-nano crystal is difficult to implement, and the double-sided adhesive tape has the problem of physical and chemical compatibility with a plurality of protein crystallization solutions; furthermore, this technical solution has no description about the layout scheme of the protein solution and its equilibrium solution/precipitation solution, that is, it is not clear what crystallization method is adopted, such as vapor diffusion, liquid-liquid diffusion or solution preparation method. The disclosed invention 'diffraction method of biomacromolecule crystal in near physiological state' (publication number CN108732193A) does not give a technical implementation principle schematic diagram, the content is similar to CN108593689A, and the method is an in-situ diffraction method without fishing out crystal, and is far from the 'near physiological state' in which various biomacromolecules coexist. The published invention "a serial crystal sample transport device and method" (publication number CN109490343A) describes an electrically rotating circular protein crystal transport box with an annular groove inside to hold the crystals and perform diffraction, which is not an in-situ diffraction technique that still requires the transfer of protein crystals from the growth solution into the box; the invention does not relate to other matching technologies.
With a conventional crystallization plate, the crystals move and the equilibration and proteinous liquids flow, which can cause the diffraction experiment to fail. In addition, the width of the existing commercial crystallization plate exceeds the allowable size of the existing standard X-ray diffractometer, so that the crystallization plate needs to be subjected to diffraction twice after being installed, and the crystallization plate needs to be turned upside down in the middle.
Therefore, the development of new devices and crystallization plates/cassettes became the key to solving the above problems.
Disclosure of Invention
In order to solve the problems and the defects, the patent discloses a device for protein crystal in-situ X-ray diffraction, which can be used for inspecting and positioning the protein crystal in a crystallization chamber. The device consists of a disc-shaped crystallization box, a three-degree-of-freedom inspection positioning mechanism with two-dimensional translation and one-dimensional rotation, an illuminating mechanism, a microscopic observation imaging mechanism, an X-ray absorption mechanism and a connecting and supporting mechanism. The inspection positioning mechanism controls the disc-shaped crystallization box to realize two-dimensional translation positioning, and the inspection motor drives the crystallization box to rotate for inspection; under the unidirectional or bidirectional illumination of the illumination mechanism, the protein crystal is found through a long-focus microscope, and the X-ray accurately irradiates the crystal by virtue of the fine positioning adjustment of the routing inspection positioning mechanism; x-rays that travel straight after passing through the crystal are absorbed by the absorbing mechanism. Wherein, the discoid crystallization box need not to incline when earlier stage growth protein crystal and installs on the device, and the level is placed in the constant temperature incubator usually, is the state of inclining after adorning on the device, because of having taken fixed measure to albumen liquid, equilibrium liquid and protein crystal in the crystallization box, consequently can avoid their flow or removal and influence the diffraction experiment.
The two-dimensional translation positioning device is suitable for directly fixing a crystallization box after crystal growth is finished, and two-dimensional translation positioning is carried out on the crystallization box in a vertical plane by means of two mutually vertical slide rails; the crystallization tanks on the crystallization boxes are distributed in a circumferential equidistant manner, and as long as one crystallization tank is positioned, all the crystallization tanks on the same circumference can be detected and diffracted through the rotation of the crystallization boxes, so that the trouble that some crystallization boxes need to be turned over in the half-range of diffraction is avoided; two sets of illuminating mechanisms are irradiated and transmitted, and illuminating light sources with various wavelengths are used, so that the protein crystal is observed, identified and positioned more conveniently and efficiently; the X-ray absorption nozzles with different specifications and a plurality of transmission light sources are arranged on the same frame and can be automatically selected and replaced according to the size of the protein crystal, so that the quality of diffraction data is improved; in the crystallization tank of the crystallization chamber, the gelled protein liquid, the equilibrium liquid, the porous material absorption equilibrium liquid, the adsorption layer fixed crystal and the like are used to effectively fix the gelled protein liquid, the equilibrium liquid, the porous material absorption equilibrium liquid and the adsorption layer fixed crystal, so that the interference of diffraction experiments is avoided. In addition, the innovative invention can avoid the risk of damaging the crystals due to complex operations such as fishing, liquid nitrogen freezing, transferring, liquid nitrogen storage and the like of the micro protein crystals, and the development and application of the device can have considerable economic and social benefits if the capital cost, the time cost and the labor cost of the preparation and purification of the protein and reagent consumables in the early stage are considered.
Drawings
FIG. 1 is a schematic view of the apparatus of the present invention;
FIG. 2 is a schematic view of the cross-sectional structure A-A in FIG. 1;
FIG. 3 is a schematic view of a first design of a composite stent;
FIG. 4 is a schematic view of a second design of a composite stent;
FIG. 5 is a schematic view of a microscope stand component layout;
FIG. 6 is a schematic diagram of an orthographic projection structure of a crystallization box;
FIG. 7 is an orthographic view of the work orientation of the crystallization box and the composite support;
FIG. 8 is a side projection view of the crystallization box and the composite support in the working position.
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
The device comprises an inspection positioning mechanism, a microimaging mechanism, a crystallization box mechanism, an illuminating mechanism, a diffraction absorption mechanism and a supporting mechanism, wherein the inspection positioning mechanism is a preferred example shown in figure 1 and comprises a large gear 6, a toothed belt 7, a base 8, an inspection motor 9, a cantilever 10, a small gear 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 microimaging mechanism is composed of a microscope 26 and a regulator 27 shown in figure 2, the crystallization box mechanism is composed of a crystallization box 5, a crystallization tank 31, a balance liquid 32, a protein liquid 33 and a protein crystal 34 shown in figures 6, 7 and 8, the illuminating mechanism is composed of a transmission illuminating light source composed of a G lamp 19, a B lamp 21 and an R lamp 23 shown in figures 2 and 3, a lamp holder 24, a blue light 35 emitted by the blue light 28, a green light 36 emitted by the green light lamp 29, a red light 37 emitted by the red light 30 and a white light 38 emitted by the microscope 26, the illuminating mechanism is composed of a falling illuminating light source which is distributed and irradiates on the same protein liquid 25 and the same protein liquid, the base 33, the fluorescent lamp 25, the diffraction absorption mechanism is composed of a base 20, the microscope base 20, the diffraction absorption mechanism, the microscope base 20 and the diffraction absorption mechanism, the microscope base 20.
2. Principle of operation
Before the whole device is started to perform in-situ X-ray diffraction by mounting the crystallization box 5 on the gear shaft 16, a key preparatory work is required, namely, protein crystals 34 are grown, a balance liquid 32 and a protein liquid 33 are added into a crystallization tank 31 on the crystallization box 5, the crystallization box 5 is flatly placed in a constant temperature incubator after sealing, and after the expected crystal growth is completed, the crystallization box 5 is taken out and is mounted on the gear shaft 16 in a side-standing manner. In which the movement of the equilibration fluid 32, the protein fluid 33 and the protein crystals 34 is avoided, see the above section "summary of the invention".
The X motor 14 and the Y motor 15 respectively drive the X slide rail 12 and the Y slide rail 13, the inspection motor 9 is matched to drive the crystallization box 5 to rotate through the pinion 11, the toothed belt 7 and the bull gear 6, protein liquid 33 in a certain designated crystallization tank 31 is adjusted to the visual field of the microscope 26, and after an object which is possibly protein liquid 34 is found, illumination can be changed for further judgment.
The inspection positioning mechanism is further finely adjusted so that the protein crystal 34 is just under the irradiation of the X-ray 25. On the other hand, the diffraction absorption mechanism is adjusted, and an appropriate absorption nozzle, such as the S absorption nozzle 18, is selected according to the size of the protein crystal 34.
After the experiment of a pond is completed, the inspection motor 9 drives the crystallization box 5 to inspect other crystallization ponds located at the same circumferential position on the crystallization box 5. After the cell experiment on the same circumference is completed, the crystallization cells on the other circumference can be brought into the field of view of the microscope 26 by using only the Y slide. This is done until all is done.
Certain specific details are set forth below in connection with the description of the construction of various components.
3. Structure of each part
The bottom plate 1 provides a mounting platform for other mechanisms, is fixed on a working table of an X-ray diffractometer through bolts, is provided with M6 holes and phi 6 holes which are distributed in an array, and the central moment of the adjacent holes is 25 mm. The material is metal, preferably duralumin. The length of the bottom plate 1 is 200-500mm, and the width is 150-300 mm.
The base 2 is used for supporting the diffraction absorption mechanism and part of the illumination mechanism, such as the composite support 3 and the lamp nozzle motor 17, and can be in a simple cuboid shape, and the upper part can also be designed into a semi-cylindrical surface. The base 2 has the following dimensions: the length is 100-200mm, the width is 50-100mm, and the height is 200-400 mm. The metal material is preferably duralumin.
The composite bracket 3 is used for mounting a multicolor lighting lamp and absorption nozzles with different specifications, the preferred structure is shown in figure 3, six arms are distributed at equal angles, the structure can be improved to the structure shown in figure 4 in consideration of the rigidity of the bracket, the number of the arms is not limited to six, and the arms can be fewer or more, such as four or eight, in the embodiment, an S absorption nozzle 18, a G lamp 19, an M absorption nozzle 20, a B lamp 21, an L absorption nozzle 22 and an R lamp 23 are sequentially arranged at the end parts of the six arms of the composite bracket 3 and can be connected through bolts or be adsorbed through tenon-and-mortise structures or magnetic force, the length of the arms of the composite bracket 3 is 30-120mm, the width is 3-10mm, the thickness is 0.3-1mm, the material is preferably metal, the material is preferably tool steel, the width of the rings in the structure shown in figure 4 is 3-10mm, the thickness is 0.1-0.5mm, the material is preferably metal or organic material, the tool steel is preferably metal, and the other parts are preferably brass.
The microscope base 4 is a support for the microscopic imaging mechanism and part of the illumination mechanism, and in this embodiment, it forms a rectangular parallelepiped, the upper half is processed with inclined holes distributed in a conical surface for installing the lamp holder 24, the microscope 26 and the adjuster 27, the two inclined holes have a hole for passing the X-ray 25 at the symmetrical axis position, the diameter of the hole is 3-10mm, and fig. 5 is a front view of the component. The microscope base 4 is arranged on the bottom plate 1 through bolts, and can also be directly arranged on the working table of the X-ray diffractometer, the height is 200-400mm, the width is 50-150mm, and the thickness is 50-100 mm. The metal material is preferably duralumin.
The crystallization cassette 5 is used for growing high quality protein crystals and can be mounted on a gear shaft 16 and rotationally moved in a vertical plane to perform inspection. The crystallization box 5 is a disk structure, and the crystallization tanks 31 are arranged on the disk structure in a concentric circle multi-circle array, as shown in fig. 6, the structural design enables the three-degree-of-freedom inspection positioning mechanism to conveniently position each crystallization tank on the light path of the X-ray 25. The thickness of the crystallization box 5 is 5-15mm, the diameter is 100-200mm, the diameter of the central mounting hole is 5-10mm, the material is transparent high molecular material, and the injection molding is carried out.
The large gear 6 is coaxial with the crystallization box 5 for transmitting the rotation of the small gear 11 to the crystallization box 5 through the toothed belt 7. The diameter of the big gear 6 is 15-30mm, the thickness is 3-10mm, and the big gear is made of metal or high polymer material, preferably brass.
The toothed belt 7 is used to transmit the rotation of the pinion 11 to the gearwheel 6, the specifications of which are matched to the gearwheels for a standardized commercial product.
The base 8 is used for mounting and positioning the gear shaft 16, and is structurally shown in fig. 1 and 2, the height is 80-150mm, the width of a mounting surface is 30-100mm, the thickness is 5-10mm, and the base is made of metal materials, preferably duralumin.
The inspection motor 9 is used for providing power for the rotation of the crystallization chamber, and is preferably a stepping motor, the rotating speed is 30-120rpm, and the stepping angle is 0.05-1 degree.
The pinion 11 is used for transmitting the rotary motion of the inspection motor 9 to the bull gear 6 through the toothed belt 7 and further to the crystallization box 5. The diameter of the pinion 11 is 8-20mm, the thickness is 3-10mm, and the pinion is made of metal or high polymer material, preferably brass.
The X slide rail 12 is used for adjusting and positioning the transverse position of the crystallization box 5 in a plane parallel to the paper surface, and is of a stepping motor and lead screw structure, the minimum displacement is 0.005mm, the repeated positioning precision is 0.01mm, the length of the slide rail is 200-400mm, and the stroke is not less than 50 mm.
The Y slide rail 13 is used for adjusting and positioning the longitudinal position of the crystallization box 5 in a plane parallel to the paper surface, and is of a stepping motor and lead screw structure, the minimum displacement is 0.005mm, the repeated positioning precision is 0.01mm, the slide rail length is 200-400mm, and the stroke is not less than 100 mm.
The X motor 14 is used for driving the X slide rail 12 to move, and is preferably a stepping motor, the rotating speed is 60-180rpm, and the stepping angle is 0.1-2 degrees.
The Y motor 15 is used for driving the Y slide rail 13 to move, and is preferably a stepping motor, the rotating speed is 60-180rpm, and the stepping angle is 0.1-2 degrees.
The gear shaft 16 is used for mounting the crystallization box 5 and the large gear 6 on the base 8, has a diameter of 4-10mm, and is made of metal, preferably brass.
The lamp nozzle motor 17 is used for driving the composite bracket 3 to rotate to realize the replacement of the illuminating lamp and the absorbing nozzle, and preferably a stepping motor with the rotating speed of 30-120rpm and the stepping angle of 0.05-1 degree.
The S absorption nozzle 18 is a metal block having a small diameter and absorbing the X-ray 25, and is cylindrical, and one end thereof is formed with a conical recess. The diameter of the S absorption nozzle 18 is 0.1-0.5mm, the length is 5-15mm, and the material is lead alloy or tungsten alloy.
The G lamp 19 is used as a medium wavelength visible light illuminating device for transmitting illumination to the crystallization tank 31, and is usually green light, and may be replaced by a white light lamp, preferably L ED lamp, with a power of 0.05-0.5W.
The M absorption nozzle 20 is a metal block having a middle diameter and absorbing X-rays 25, and is cylindrical, and a conical recess is formed at one end. The M absorption nozzle 20 has a diameter of 0.5-1.0mm and a length of 5-15mm, and is made of lead alloy or tungsten alloy.
The B lamp 21 is an illumination device for transmitting a short wavelength visible light for illuminating the crystal cell 31, and is usually a blue light lamp, and may be replaced with a white light lamp, preferably an L ED lamp with a power of 0.05-0.5W.
L the absorption nozzle 22 is a metal block with large diameter for absorbing X-ray 25, is cylindrical, and has a conical pit at one end, L the 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 illumination device for transmitting the longer wavelength visible light for illuminating the crystallization tank 31, and is usually a red light emitting lamp, and may be replaced with a white light lamp, preferably an L ED lamp, with a power of 0.05-0.5W.
The lamp holder 24 is used for mounting and supplying power to the blue light lamp 28, the green light lamp 29 and the red light lamp 30, and is cylindrical, 10-30mm in diameter, 50-100mm in length and made of metal.
The X-ray 25 is not an essential element of the invention and is added for convenience in describing the working principle of the device, it is not a solid element but a beam of invisible light, with a wavelength between 0.1 and 0.2nm and a beam diameter of 0.01 to 0.5 mm. X-rays are harmful to human bodies and safety protection is needed when people approach.
The microscope 26 is used for searching and observing the protein crystal 34 in the crystallization pool 31, the focal length is 100-300mm, the physical magnification is 50 times at most, and the diameter is 10-30mm with white light coaxial illumination.
The blue light lamp 28, when energized, emits blue light 35 to illuminate the protein crystal 34 in an epi-illumination mode, preferably a single color L ED, with a power of 0.05-0.5W.
The green light lamp 29 is energized to emit green light 35 for epi-illumination of the protein crystal 34. preferably, a single color L ED with a power of 0.05-0.5W.
The red light lamp 30 emits red light 35 after being electrified, and is used for illuminating the protein crystal 34 in an epi-illumination mode, wherein the red light lamp is preferably single-color L ED and has the power of 0.05-0.5W.
The crystallization tank 31 and the small tank for growing the protein crystal 34 on the crystallization box 5 have openings in the shape of an isosceles trapezoid with a short centripetal edge and a long opposite edge, wherein the small tank is filled with the protein liquid 33 and the balance liquid 32, the structure is shown in figures 7 and 8, the inner part of the small tank is in a step structure, the steps are arranged on the outer peripheral side, the balance liquid 32 is arranged in the space below the steps, the protein liquid 33 is arranged in the pits on the steps, the number of the pits is 1-3, the pits are cylindrical or spherical, the depth is 1-3mm, and the opening diameter is 2-4 mm. The material is the same as the crystallization box 5 and is integrated by injection molding. The crystallizing tanks 31 are distributed on the crystallizing box 5 in concentric circumference equidistant array, the depth of the tanks 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 3-5 mm.
The equilibrium liquid 32 is an aqueous solution having a vapor pressure lower than that of the protein liquid 33, and absorbs the vapor evaporated from the protein liquid 33.
The protein solution 33 is an aqueous solution in which highly pure active protein is dissolved, and usually contains a chemical agent to buffer pH change. The water in the protein liquid 33 is evaporated into vapor and absorbed by the equilibrium liquid 32 to reach supersaturation, and finally, protein crystals 34 are separated out and grow gradually.
The protein crystal 34 is a new phase in which the protein in the protein liquid 33 reaches a supersaturated state due to the continuous evaporation of the solvent water and is precipitated in the form of a solid with orderly arranged molecules.
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 (10)
1. The utility model provides a device for protein crystal normal position X-ray diffraction, including X slide rail (12) by X motor (14) drive, Y slide rail (13) of Y motor (15) drive and by installing patrolling and examining motor (9) on cantilever (10) through pinion (11), the drive that tooth belt (7) and install gear wheel (6) on gear shaft (16) constitute patrols and examines positioning mechanism, and by bottom plate (1), base (2), microscope base (4) and the supporting mechanism that base (8) are constituteed, a serial communication port, the device still includes by disc crystallization box (5), crystallization tank (31), balanced liquid (32), the crystallization chamber mechanism that albumen liquid (33) and albumen crystal (34) are constituteed jointly, and be used for observing the microscope (26) of discerning the crystal and the microimaging mechanism that its regulator (27) are constituteed, by G lamp (19), B lamp (21), R lamp (23), lamp stand (24), blue light lamp (28), green light lamp (29), the illumination mechanism that red light lamp (30) are constituteed, by compound support (3), L) absorb motor (17), the absorption mechanism is constituteed, X ray mouth (20) and absorption mechanism.
2. The apparatus for in situ X-ray diffraction of protein crystals as claimed in claim 1, wherein the blue light (35) emitted from the blue light lamp (28), the green light (36) emitted from the green light lamp (29), the red light (37) emitted from the red light lamp (30) and the white light (38) emitted from the microscope (26) are distributed in a focus shape and are irradiated on the same protein liquid (33) and protein crystal (34) together with the X-ray (25).
3. The device for in-situ X-ray diffraction of protein crystals as claimed in claim 1, wherein the disc-shaped crystallization box (5) is mounted on a gear shaft (16) through a middle mounting hole and can be driven by a routing inspection motor (9) to rotate in a vertical plane through power transmitted by a pinion (11), a toothed belt (7), a gearwheel (6) and the gear shaft (16).
4. The apparatus for in situ X-ray diffraction of protein crystals as claimed in claim 1, wherein the illumination mechanism is equipped with both epi-and transmission multicolor illumination, the G lamp (19), the B lamp (21) and the R lamp (23) are transmission illumination sources, and the blue light (35) emitted from the blue lamp (28), the green light (36) emitted from the green lamp (29), the red light (37) emitted from the red lamp (30) and the white light (38) emitted from the microscope (26) are epi-illumination sources.
5. The device for in-situ X-ray diffraction of protein crystals as claimed in claim 1, wherein the end of the arm of the composite support (3) is simultaneously provided with a G lamp (19), a B lamp (21) and an R lamp (23) for illumination, and an S absorption nozzle (18), an M absorption nozzle (20) and an L absorption nozzle (22) for absorbing X-rays (25), and the composite support (3) is driven by a nozzle motor (17) to rotate, so that the illumination lamp and the absorption nozzles can be replaced.
6. The device for in-situ X-ray diffraction of protein crystals as claimed in claim 3, wherein the crystallization box (5) is in a disc shape, the crystallization tanks (31) are arranged in a concentric circumferential array, and the protein liquid (33) is positioned on the step at the outer side inside 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 5-10 mm; the opening of the crystallization tank (31) is in an isosceles trapezoid shape with a centripetal edge short and an opposite side length, 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 3-5 mm.
7. The device for in-situ X-ray diffraction of protein crystals as claimed in claim 5, wherein the six arms of the composite support (3) are radially distributed with equal included angles, the ends of the arms are connected into a whole by a ring, 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 as claimed in claim 5, wherein the number of the arms of the composite support (3) is more than six, the arms are distributed in a radial shape with equal included angles, the tail ends of the arms are connected into a whole by a ring, the arms are 30-120mm long, 3-10mm wide and 0.3-1mm thick, and are made of metal.
9. The device for in-situ X-ray diffraction of protein crystals as claimed in claim 5, wherein the number of the arms of the composite support (3) is less than six, the arms are distributed in a radial shape with equal included angles, the ends of the arms are connected into a whole by a ring, the arms are 30-120mm long, 3-10mm wide and 0.3-1mm thick, and the material is metal.
10. The device for in-situ X-ray diffraction of protein crystals as claimed in any one of claims 7 to 9, wherein the arms of the composite support (3) are made of tool steel.
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| CN201910666162.1A CN110361407B (en) | 2019-07-23 | 2019-07-23 | Device for protein crystal in-situ X-ray diffraction |
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| CN201910666162.1A CN110361407B (en) | 2019-07-23 | 2019-07-23 | Device for protein crystal in-situ X-ray diffraction |
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