CN110095485B - Preparation and structure analysis method of protein tiny crystal frozen sample - Google Patents
Preparation and structure analysis method of protein tiny crystal frozen sample Download PDFInfo
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- CN110095485B CN110095485B CN201810081566.XA CN201810081566A CN110095485B CN 110095485 B CN110095485 B CN 110095485B CN 201810081566 A CN201810081566 A CN 201810081566A CN 110095485 B CN110095485 B CN 110095485B
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- 239000013078 crystal Substances 0.000 title claims abstract description 102
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 46
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 46
- 239000012520 frozen sample Substances 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims description 15
- 238000003696 structure analysis method Methods 0.000 title description 4
- 239000000523 sample Substances 0.000 claims abstract description 60
- 238000000034 method Methods 0.000 claims abstract description 48
- 238000013480 data collection Methods 0.000 claims abstract description 13
- 238000012216 screening Methods 0.000 claims abstract description 9
- 238000004458 analytical method Methods 0.000 claims abstract description 8
- 238000012545 processing Methods 0.000 claims abstract description 5
- 238000012916 structural analysis Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 23
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 22
- 229910052802 copper Inorganic materials 0.000 claims description 22
- 239000010949 copper Substances 0.000 claims description 22
- 239000013081 microcrystal Substances 0.000 claims description 13
- 230000008014 freezing Effects 0.000 claims description 10
- 238000007710 freezing Methods 0.000 claims description 10
- 238000005286 illumination Methods 0.000 claims description 10
- 238000011068 loading method Methods 0.000 claims description 9
- 229920001223 polyethylene glycol Polymers 0.000 claims description 9
- 230000035484 reaction time Effects 0.000 claims description 8
- 239000012488 sample solution Substances 0.000 claims description 8
- 238000009210 therapy by ultrasound Methods 0.000 claims description 7
- 230000001276 controlling effect Effects 0.000 claims description 6
- 229920000604 Polyethylene Glycol 200 Polymers 0.000 claims description 5
- 239000006185 dispersion Substances 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 4
- 239000011549 crystallization solution Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 229910000629 Rh alloy Inorganic materials 0.000 claims description 2
- HNWNJTQIXVJQEH-UHFFFAOYSA-N copper rhodium Chemical compound [Cu].[Rh] HNWNJTQIXVJQEH-UHFFFAOYSA-N 0.000 claims description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 2
- 229910052737 gold Inorganic materials 0.000 claims description 2
- 239000010931 gold Substances 0.000 claims description 2
- 238000005360 mashing Methods 0.000 claims description 2
- 229910052759 nickel Inorganic materials 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000010008 shearing Methods 0.000 claims description 2
- 238000002635 electroconvulsive therapy Methods 0.000 claims 1
- 238000002003 electron diffraction Methods 0.000 abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 238000010521 absorption reaction Methods 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000002441 X-ray diffraction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 238000002524 electron diffraction data Methods 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 108010048112 Amyloidogenic Proteins Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 238000012866 crystallographic experiment Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 210000004896 polypeptide structure Anatomy 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 102000010838 rac1 GTP Binding Protein Human genes 0.000 description 1
- 108010062302 rac1 GTP Binding Protein Proteins 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000001665 trituration Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
-
- 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
-
- 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
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- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Analysing Materials By The Use Of Radiation (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention provides an analysis method of a protein tiny crystal structure. Specifically, the method comprises the steps of: sample processing, preparing frozen samples, crystal screening, data collection and structural analysis. The method analyzes the structure of the protein tiny crystal by a freeze electron microscope electron diffraction method, and the resolution of the analyzed structureResolution is more resolved
Description
Technical Field
The invention belongs to the field of structural biology, and particularly relates to a method for analyzing a protein micro-crystal structure by a freezing electron microscope.
Background
Protein crystal structure analysis with X-ray diffraction technique as core is important to explain physiological and pathological functions of protein. The crystal scale of conventional crystallographic studies is in the tens to hundreds of microns/dimension. However, many important proteins, such as pathogenic amyloid and membrane proteins in neurodegenerative diseases, can only form ultra-microcrystals of tens of nanometers to several micrometers due to the influence of various factors such as flexibility, heterogeneity and the like, and the atomic resolution structure of the micro crystals cannot be resolved by using a conventional X-ray diffraction technology, so that the invention provides a freeze electron microscopy electron diffraction method which is feasible for the crystals so as to resolve the atomic resolution structure of the micro crystals. However, the microcrystalline electron diffraction technique is a novel technique, and there is no method for preparing and analyzing the structure of a mature sample of tiny crystals of a protein.
In view of the foregoing, there is a strong need in the art to develop a method for sample preparation and structural analysis of protein crystals; in particular to a method for preparing a sample and analyzing a structure of a tiny crystal.
Disclosure of Invention
The invention aims to provide a method for preparing a sample and analyzing a structure of a protein crystal; in particular to a method for preparing a sample and analyzing a structure of a tiny crystal.
In a first aspect of the present invention, there is provided a method for analyzing a protein micro-crystal structure, comprising the steps of:
(1) Processing a protein micro-crystal sample to obtain a sample liquid, wherein the sample liquid is a solution containing dispersed protein crystals;
(2) Preparation of frozen samples: freezing the sample loading liquid to obtain a frozen sample;
(3) And (3) crystal screening: searching a crystal for analyzing a structure through an electron microscope, and determining the position of the crystal and analysis conditions of the electron microscope;
(4) And (3) data collection: collecting diffraction images of different angles of the crystal;
(5) Structural analysis: and analyzing and obtaining the structure of the protein crystal according to diffraction images of different angles.
In another preferred embodiment, the structure of the protein crystals is resolvedPreferably, the +>
In another preferred embodiment, the structure of the resolved protein microcrystals (thickness of less than 200nm, length and width as large as possible) isPreferably, the +>
In another preferred example, the protein tiny crystals are crystals with the thickness less than or equal to 200nm.
In another preferred embodiment, the analysis conditions include: the size of the aperture (preferably 10 μm), the intensity of the illumination (preferably)。
In another preferred embodiment, the analysis conditions further include: and (3) moving the single crystal to the position of the selective diaphragm, and adjusting the height of the sample stage so that the crystal cannot move out of the selective diaphragm in the rotation process of the sample stage.
In another preferred embodiment, in step (2), the method further comprises the steps of:
(2.1) providing a carrier network;
(2.2) applying a sample solution (preferably 2-5 μl) to the surface of the carrier web;
(2.3) removing the liquid to allow the protein crystals to settle on said carrier web;
(2.4) carrying out quick freezing on the protein crystals on the carrier net and the surface to obtain a frozen sample.
In another preferred embodiment, the protein crystals in the frozen sample have a thickness of 200nm or less.
In another preferred embodiment, in the step (2.3), further comprising
(2.3.1) drawing liquid from the back side of the carrier web such that the front side of the carrier web leaves behind a viscous liquid containing protein crystals;
(2.3.2) washing said carrier web with 2-10% PEG solution and blotting said PEG solution from the back of the carrier web;
(2.3.3) the remaining liquid was sucked off with an instrument, leaving protein crystals on the front side of the carrier web.
In another preferred embodiment, in the step (2.1), the carrier web is a hydrophilized carrier web.
In another preferred embodiment, in step (2.1), the carrier web is selected from the group consisting of: copper mesh, gold mesh, nickel mesh, or copper rhodium alloy mesh; preferably, the carrier net is a copper net.
In another preferred embodiment, in the step (2.1), the specification of the carrier net is R2/2.
In another preferred embodiment, in the step (2.1), the carrier mesh is a 400 mesh copper mesh.
In another preferred embodiment, in step (2.2), 1 to 8 μl of sample is added to the carrier web; preferably, 2 to 5. Mu.l of sample is added to the carrier web.
In another preferred embodiment, after said step (2.3), step (2.2) is repeated until sufficient protein crystals remain on the carrier network.
In another preferred embodiment, in step (2.4), the frozen sample is obtained by inserting the carrier web into liquid ethane for freezing.
In another preferred embodiment, in step (2.3.2), the PEG solution is a 1-20wt% PEG solution, preferably a 1-10% (w/t) (e.g., 5%) PEG solution.
In another preferred embodiment, in the step (2.3.2), the PEG solution is a PEG200 solution.
In another preferred embodiment, in the step (1), the treatment further comprises the steps of:
(1.1 a) mashing the crystal nuclei to uniformly disperse the crystals in the droplets to obtain a dispersion of crystals in the pool;
(1.2 a) transferring the crystals together with the pool to a centrifuge tube.
In another preferred embodiment, in step (1.1 a), the trituration is performed by a needle-like tool.
In another preferred embodiment, the process further comprises the steps of:
(1.3 a) dilution of the dispersion (dilution based on crystal size, dispersibility, etc. to give an observable sample).
In another preferred embodiment, in step (1), for larger crystal (micron-scale or hundreds of microns) proteins, the process further comprises the steps of:
(1.1 b) shearing off the pipette tip and transferring the protein crystal sample and crystallization solution into the ep tube;
(1.2 b) subjecting said protein crystal sample solution to sonication and/or shaking;
(1.3 b) precipitating large crystals, and taking the upper liquid as the loading liquid.
In another preferred embodiment, in step (1.2 b), the ultrasonic treatment is water bath ultrasonic.
In another preferred embodiment, in the step (1.2 b), the ultrasonic power of the ultrasonic treatment is 10% -20%.
In another preferred embodiment, in the step (1.2 b), the time of the ultrasonic treatment is 0.1 to 1s; preferably 0.4 to 0.8s.
In another preferred embodiment, in the step (1.2 b), the shaking treatment is vortex shaking.
In another preferred embodiment, in the step (1.2 b), the shaking treatment is performed 2 to 3 times.
In another preferred embodiment, in step (1.3 b) large crystals are precipitated by spin-drying.
In another preferred example, in the step (3), the electron microscope is a 120KV frozen electron microscope provided with a field selection diaphragm.
In another preferred embodiment, in the step (3), the illumination intensity of the electron microscope isPreferably +.>
In another preferred embodiment, in step (4), the diffraction image collection includes: and controlling the sample table to rotate at a constant speed, controlling the camera to synchronously expose, and then collecting diffraction images.
In another preferred embodiment, in step (4), the sample stage is tilted at a constant speed in synchronization with the camera exposure.
In another preferred embodiment, the data collection is performed using a FEI Tecnai 200Kv electron microscope equipped with a FEG filament.
In another preferred embodiment, the data collection is performed by using a LaB-equipped device 6 Is performed by a 120Kv electron microscope.
In another preferred embodiment, the diffraction image collection further comprises:
measuring the reaction time of a camera and the reaction time of a sample stage;
setting an initial rotation angle and an end angle, and setting the rotation speed step length of the sample stage and the corresponding exposure time;
the height of the copper net is regulated, so that the frozen sample is always positioned on the selective shaft in the tilting process of the sample table;
the sample stage was tilted to the starting angle and data collection was started.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 Structure (resolution) of RAC1 polypeptide sysgys resolved by the method of the present invention)
FIG. 2 Structure (resolution) of a conventional protein lysozyme polypeptide sysgys resolved by the method of the present invention)。
Detailed Description
The present inventors have studied intensively for a long time and developed a method for preparing a frozen sample of a mature protein crystal for a frozen electron microscope and a method for analyzing the crystal structure of the protein according to data obtained by the electron microscope. According to the characteristics of protein nano-scale micro crystals, a whole set of crystal screening, electron microscope sample preparation, data collection and structure analysis methods are developed. And resolving the ultra-high resolution protein structure approaching 1 angstrom by using a 120kv electron microscope for the first time.
Sample processing method
The amyloid aggregates the core polypeptide to form tiny crystals mostly in the shape of needle crystals, and many of the tiny crystals are sea urchin-shaped, and a large number of the needle crystals aggregate into clusters, so that the tiny crystals are difficult to process to obtain dispersed single crystals. Because the clustered crystals are visible under an optical microscope, the intermediate nuclei are first mashed with a needle-like tool to allow uniform dispersion of the needle-like crystals in the droplets, and then the crystals are sucked into a centrifuge tube together with the pool liquid and diluted appropriately to give a suspension of crystals for frozen sample preparation.
For larger crystals, an ultrasonic or vortex vibration treatment is required. The appropriate amount of sample and crystallization solution was transferred to an ep tube of appropriate size. Carrying out water bath ultrasonic treatment: the ultrasonic wave is carried out in a water bath ultrasonic device which is regulated to the lowest power, the time is controlled within 1s, and the integrity of the crystal package is not damaged as much as possible. Vortex vibration: the swirling time is 5s, and if the microcrystals are concentrated, a plurality of swirling times are needed.
Frozen sample preparation
The loading was determined based on the concentration of the crystals, typically in a volume of 4 μl, and samples were prepared for manual as well as vitro combinations. Firstly, a proper amount of sample is loaded on a frozen copper net after hydrophilization treatment, a copper net with the model number of 2/2 and 400 meshes of Quantifoil company is generally used, and after the sample is loaded, the liquid is immediately sucked by filter paper on the other side, so that crystals are settled on the copper net. If the crystal concentration is low, the sample can be applied for multiple times and sucked dry on the back surface of the copper mesh. Since the crystal needs to use a precipitant in the growth process, the solution is viscous, so that the solution cannot be completely absorbed in the vitro bot, a thicker ice layer is generated on the copper mesh, electrons cannot penetrate, a good diffraction pattern cannot be obtained or the diffraction quality is poor, and the solution needs to be removed as much as possible in the sample preparation process. Based on this, we invented that after the liquid is sucked off, 4. Mu.l of 5% PEG200 solution is used for washing, and then sucked off from the back of the copper mesh, so that the dual effects of removing the viscous solution of the sample and preventing freezing of crystals are achieved. Finally, the water absorption time is set to 30s by using a Vitrobot, the water absorption times are 2 times, the water absorption pressure is 1, and the residual solution is completely absorbed. The copper mesh was rapidly inserted into liquid ethane for rapid freezing and stored in a liquid nitrogen environment.
Crystal screening
After the sample preparation is completed, a 120KV cryoelectron diffraction screen equipped with a field diaphragm is used for microcrystalline electron diffraction screening. The method is performed in a low dose mode by using a cryo-electron microscope. The Search option is tuned to LM image mode with a magnification of around 100 x for determining the area where crystals are present. The Focus option is turned to the diffraction mode and the camera length is one step greater than the Exposure. The Focus option is used to determine the size of the crystal location and the placement of the selection field stop. Adjusting the Exposure option to be in a diffractation mode, carrying out axis combination under the option, adjusting the light intensity and the illumination area, adjusting the option to be spot size and intensity, and finally enabling the illumination intensity to be the same as the illumination intensityThe light is then focused to a small point using the focus knob while the field stop and beam stop are determined to be centered on the optical axis. After the adjustment under the Exposure option is completed, the parameters such as intensity, which are related to the light intensity, cannot be adjusted again under the Search and Focus options.
After the electron microscope is regulated, selecting an area with a diaphragm under a Search option, switching to a Focus option, specifically selecting a crystal to be diffracted, loading a diaphragm with a proper size for selecting an area, placing a beam stop, switching to an Exposure option, directly exposing, and setting the Exposure time to follow the diffraction intensity of a sample.
Data collection
Microcrystalline electron diffraction data collection was performed using software. And controlling the sample table to tilt at a constant speed and synchronously exposing the camera, firstly measuring the reaction time of the camera and the reaction time of the sample table, and then setting the initial rotation angle and the end angle, the rotation speed step length of the sample table and the corresponding exposure time. After the setting is finished, the height of the copper mesh is adjusted, so that the crystal to be diffracted is always positioned on the selective area shaft in the tilting process of the sample table, then the sample table is tilted to an initial angle, the selective area diaphragm and the beam stop are loaded, the file storage name and the storage address are set, and data collection is started.
Structural resolution
The collected data file is in the. mrc format, which is first converted to an. Img format file based on the measured camera length. The format file can directly utilize X-ray crystallography software xds to process data, integration and scale to obtain a density mtz file with missing phase information. And using Phenix software to make Molecular replace to obtain phase information, and further refining to finally analyze the structure of the micro crystal.
The main advantages of the invention include:
the invention provides a structure analysis method of tiny crystals with the thickness smaller than 200nm, which fills the blank of the field. The protein structure resolved by the method has high resolution, and achieves the aim of larger crystalsIs greater than the resolution of protein micro-crystals by +.>The following resolution.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Example 1
Structural resolution of polypeptide SYSGYS
1) Sample processing
The intermediate nuclei are first mashed with a needle-like tool to allow uniform dispersion of the needle-like crystals in the droplets, and then the crystals are sucked into a centrifuge tube together with the pool liquid and appropriately diluted to obtain a suspension of crystals for frozen sample preparation.
2) Frozen sample preparation
The loading was determined based on the concentration of crystals and the volume was 4 μl and samples were prepared manually and in vitro. Firstly, a proper amount of sample is loaded on a frozen copper net after hydrophilization treatment, a copper net with the model number of 2/2 and 400 meshes of Quantifoil company is generally used, and after the sample is loaded, the liquid is immediately sucked by filter paper on the other side, so that crystals are settled on the copper net. If the crystal concentration is low, the sample can be applied for multiple times and sucked dry on the back surface of the copper mesh. After the liquid is sucked, the liquid is washed by 4 mu l of 5% PEG200 solution and then sucked from the back of the copper mesh, thereby achieving the dual effects of removing viscous solution of the sample and preventing freezing of crystals. Finally, the water absorption time is set to 30s by using a Vitrobot, the water absorption times are 2 times, the water absorption pressure is 1, and the residual solution is completely absorbed. The copper mesh was rapidly inserted into liquid ethane for rapid freezing and stored in a liquid nitrogen environment.
3) Crystal screening
After the sample preparation is completed, a 120KV cryoelectron diffraction screen equipped with a field diaphragm is used for microcrystalline electron diffraction screening. The method is performed in a low dose mode by using a cryo-electron microscope. The Search option is tuned to LM image mode with a magnification of around 100 x for determining the area where crystals are present. The Focus option is turned to the diffraction mode and the camera length is one step greater than the Exposure. The Focus option is used to determine the size of the crystal location and the placement of the selection field stop. Adjusting the Exposure option to be in a diffractation mode, carrying out axis combination under the option, adjusting the light intensity and the illumination area, adjusting the option to be spot size and intensity, and finally enabling the illumination intensity to be the same as the illumination intensityThe light is then focused to a small point using the focus knob while the field stop and beam stop are determined to be centered on the optical axis. After the adjustment under the Exposure option is completed, the parameters such as intensity, which are related to the light intensity, cannot be adjusted again under the Search and Focus options.
After the electron microscope is regulated, selecting an area with a diaphragm under a Search option, switching to a Focus option, specifically selecting a crystal to be diffracted, loading a diaphragm with a proper size for selecting an area, placing a beam stop, switching to an Exposure option, directly exposing, and setting the Exposure time to follow the diffraction intensity of a sample.
4) Data collection
Microcrystalline electron diffraction data collection was performed using software. And controlling the sample table to tilt at a constant speed and synchronously exposing the camera, firstly measuring the reaction time of the camera and the reaction time of the sample table, and then setting the initial rotation angle and the end angle, the rotation speed step length of the sample table and the corresponding exposure time. After the setting is finished, the height of the copper mesh is adjusted, so that the crystal to be diffracted is always positioned on the selective area shaft in the tilting process of the sample table, then the sample table is tilted to an initial angle, the selective area diaphragm and the beam stop are loaded, the file storage name and the storage address are set, and data collection is started.
5) Structural resolution
The collected data file is in the. mrc format, which is first converted to an. Img format file based on the measured camera length. The format file can directly utilize X-ray crystallography software xds to process data, integration and scale to obtain a density mtz file with missing phase information. And using Phenix software to make Molecular replace to obtain phase information, and further refining to finally analyze the structure of the polypeptide SYSGYS.
The analysis result is shown in FIG. 1, and the resolution of the analyzed SYSGYS polypeptide structure reaches
Example 2
Resolution of the structure of conventional crystals of lysozyme.
1) Sample processing
The appropriate amount of sample and crystallization solution was transferred to an ep tube of appropriate size. Carrying out water bath ultrasonic treatment: the ultrasonic wave is carried out in a water bath ultrasonic device with the lowest power, the ultrasonic power is 20 percent, the water bath time is 0.5s, and the integrity of the crystal package is not damaged as much as possible. And (3) sample spinning after ultrasonic treatment to enable a large block to be deposited at the bottom of the test tube, and taking the upper sample for sample preparation.
Frozen sample preparation, crystal screening, data collection, and structure resolution steps were the same as in example 1.
As shown in FIG. 2, the resolution of the structure of the conventional crystals of lysozyme precipitated was
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (14)
1. The method for analyzing the protein micro-crystal structure is characterized by comprising the following steps:
(1) Processing a protein micro-crystal sample to obtain a sample liquid, wherein the sample liquid is a solution containing dispersed protein crystals;
(2) Preparation of frozen samples: freezing the sample loading liquid to obtain a frozen sample;
(3) And (3) crystal screening: searching a crystal for analyzing a structure through an electron microscope, and determining the position of the crystal and analysis conditions of the electron microscope;
(4) And (3) data collection: collecting diffraction images of different angles of the crystal;
(5) Structural analysis: analyzing and obtaining the structure of protein crystals according to diffraction images of different angles;
in the step (2), the method further comprises the steps of:
(2.1) providing a carrier network;
(2.2) applying a sample solution to the surface of the carrier web;
(2.3) removing the liquid to allow the protein crystals to settle on said carrier web; wherein in the step (2.3), the method further comprises
(2.3.1) drawing liquid from the back side of the carrier web such that the front side of the carrier web leaves behind a viscous liquid containing protein crystals;
(2.3.2) washing said carrier web with 2-10% PEG solution and blotting said PEG solution from the back of the carrier web; wherein, PEG is PEG200; and
(2.3.3) sucking out the remaining liquid with an instrument, thereby leaving protein crystals on the front side of the carrier web;
(2.4) carrying out quick freezing on the protein crystals on the carrier net and the surface to obtain a frozen sample.
2. The method according to claim 1, wherein 2 to 5. Mu.l of the loading solution is added in the step (2.2).
3. The analytical method according to claim 2, wherein the washing is performed with 4. Mu.l of 5% PEG200 solution.
4. The method of claim 1, wherein in step (2.1), the carrier network is selected from the group consisting of: copper mesh, gold mesh, nickel mesh, or copper rhodium alloy mesh.
5. The method of claim 1, wherein in step (2.1), the carrier is a copper mesh.
6. The method of claim 1, wherein in step (1), the treatment further comprises the steps of, for the micro-crystalline protein:
(1.1 a) mashing the crystal nuclei to uniformly disperse the crystals in the droplets to obtain a dispersion of crystals in the pool;
(1.2 a) transferring the crystals together with the pool to a centrifuge tube.
7. The method of claim 1, wherein in step (1), for protein crystals on the micrometer scale, the treatment further comprises the steps of:
(1.1 b) shearing off the pipette tip and transferring the protein crystal sample and crystallization solution into the ep tube;
(1.2 b) subjecting said protein crystal sample solution to shaking treatment;
(1.3 b) precipitating large crystals, and taking the upper liquid as the loading liquid.
8. The method of claim 7, wherein the micron scale is several hundred microns.
9. The method of claim 7, wherein the shock treatment is an ultrasonic treatment.
10. The method of claim 1, wherein in step (3), the electron microscope is a 120KV cryoelectron microscope equipped with a field stop.
11. The method according to claim 1, wherein in the step (3), the illumination intensity of the electron microscope is
12. The method according to claim 1, wherein in the step (3), the illumination intensity of the electron microscope is
13. The method of claim 1, wherein in step (4), the diffraction image collection comprises: and controlling the sample table to rotate at a constant speed, controlling the camera to synchronously expose, and then collecting diffraction images.
14. The method of claim 13, wherein in step (4), the diffraction image collection further comprises:
measuring the reaction time of a camera and the reaction time of a sample stage;
setting an initial rotation angle and an end angle, and setting the rotation speed step length of the sample stage and the corresponding exposure time;
the height of the copper net is regulated, so that the frozen sample is always positioned on the selective shaft in the tilting process of the sample table;
the sample stage was tilted to the starting angle and data collection was started.
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