CN110095485A - A kind of method of the preparation and structure elucidation of protein tiny crystals frozen samples - Google Patents
A kind of method of the preparation and structure elucidation of protein tiny crystals frozen samples Download PDFInfo
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- CN110095485A CN110095485A CN201810081566.XA CN201810081566A CN110095485A CN 110095485 A CN110095485 A CN 110095485A CN 201810081566 A CN201810081566 A CN 201810081566A CN 110095485 A CN110095485 A CN 110095485A
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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 45
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 239000012520 frozen sample Substances 0.000 title claims abstract description 17
- 239000000523 sample Substances 0.000 claims abstract description 59
- 238000013480 data collection Methods 0.000 claims abstract description 13
- 238000004458 analytical method Methods 0.000 claims abstract description 10
- 238000000386 microscopy Methods 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims description 26
- 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
- 239000000243 solution Substances 0.000 claims description 22
- 239000012488 sample solution Substances 0.000 claims description 13
- 238000012545 processing Methods 0.000 claims description 12
- 230000009514 concussion Effects 0.000 claims description 7
- 239000006185 dispersion Substances 0.000 claims description 7
- 238000005286 illumination Methods 0.000 claims description 7
- 230000001360 synchronised effect Effects 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 4
- 239000013068 control sample Substances 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- 230000004044 response Effects 0.000 claims description 4
- 239000011549 crystallization solution Substances 0.000 claims description 3
- 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
- 229910052759 nickel Inorganic materials 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 238000012216 screening Methods 0.000 abstract description 6
- 238000002003 electron diffraction Methods 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 229920001223 polyethylene glycol Polymers 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
- 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
- 229920000604 Polyethylene Glycol 200 Polymers 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002604 ultrasonography Methods 0.000 description 3
- 241001400675 Sympodium Species 0.000 description 2
- 238000002441 X-ray diffraction Methods 0.000 description 2
- 239000002250 absorbent Substances 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000002528 anti-freeze Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002524 electron diffraction data Methods 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
- 230000035800 maturation Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 102000009091 Amyloidogenic Proteins Human genes 0.000 description 1
- 108010048112 Amyloidogenic Proteins Proteins 0.000 description 1
- 241000257465 Echinoidea Species 0.000 description 1
- 229910025794 LaB6 Inorganic materials 0.000 description 1
- 230000006933 amyloid-beta aggregation Effects 0.000 description 1
- -1 and volume is 4 μ l Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 238000000399 optical microscopy Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 210000004896 polypeptide structure Anatomy 0.000 description 1
- 230000008569 process Effects 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
- 238000011160 research Methods 0.000 description 1
- 238000003696 structure analysis method Methods 0.000 description 1
- 238000012360 testing method 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)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (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 present invention provides a kind of analytic methods of protein tiny crystals structure.Specifically, the method is comprising steps of sample treatment, preparation frozen samples, crystal screening, data collection and structure elucidation.The method of the present invention has passed through the structure that electron cryo-microscopy electron diffraction has parsed protein tiny crystals, the structural resolution parsedMore parse resolution ratio
Description
Technical field
The invention belongs to structure biology fields, and in particular to a kind of electron cryo-microscopy parsing protein tiny crystals structure
Method.
Background technique
It is the parsing of the protein structures of core for the physiology and pathology function of explaination protein using X-ray diffraction technology
It can be most important.The crystal scale of conventional crystalline research arrives several hundred micron/dimensions tens.But many important albumen, such as mind
Through pathogenic amyloid protein, the memebrane protein etc. in degenerative disease, due to the influence of the factors such as itself flexibility, heterogencity
The ultracrystallite that can be only formed tens nanometers to several microns can not parse such tiny crystals original using conventional x-ray diffraction technology
Sub- resolution structural, therefore invention is to the feasible electron cryo-microscopy electron diffraction of the crystalloid, to parse such tiny crystals
Atom definition structure.But crystallite electron diffraction technique is a kind of novel technology, is directed to protein there are no a kind of maturation
Tiny crystals sample preparation and structure elucidation method.
In conclusion there is an urgent need in the art to develop the method for a kind of sample preparation of protein crystal and structure elucidation；
The method of sample preparation and structure elucidation especially for tiny crystals.
Summary of the invention
It is an object of the invention to provide a kind of sample preparation of protein crystal and the methods of structure elucidation；Especially needle
The method of sample preparation and structure elucidation to tiny crystals.
In the first aspect of the present invention, a kind of analytic method of protein tiny crystals structure is provided, which is characterized in that
Include the following steps:
(1) protein tiny crystals sample is handled, obtains sample solution, the sample solution is the albumen containing dispersion
The solution of matter crystal；
(2) preparation of frozen samples: freezing processing is carried out to the sample solution, obtains frozen samples；
(3) crystal screens: finding the crystal for being used for analytic structure by Electronic Speculum, and determines the solution of crystal positions and Electronic Speculum
Analysis condition；
(4) data collection: the diffraction image of crystal different angle is collected；
(5) structure elucidation: according to the diffraction image of different angle, parsing obtains the structure of protein crystal.
In another preferred example, the structure of the protein crystal of parsingPreferably,
In another preferred example, (thickness needs to be less than 200nm to the protein tiny crystals of parsing, long and wide by big as far as possible one
Structure a bit)Preferably,
In another preferred example, the protein tiny crystals are thickness≤200nm crystal.
In another preferred example, the analysis condition include: the size (preferably 10 μm) of constituency diaphragm, illumination it is strong
Degree is (preferably)。
In another preferred example, the analysis condition further include: monocrystalline is moved on to the position of constituency diaphragm, and adjusts sample
Sample platform height, so that crystal will not remove constituency diaphragm in sample stage rotary course.
In another preferred example, it in step (2), further comprises the steps of:
(2.1) support grid is provided；
(2.2) the support grid surface of Xiang Suoshu adds sample liquid (preferably 2-5 μ l)；
(2.3) liquid is removed, falls to protein crystal in the support grid；
(2.4) protein crystal of the support grid and surface is rapidly frozen, obtains frozen samples.
In another preferred example, in the frozen samples, the protein crystal is with a thickness of≤200nm.
In another preferred example, in step (2.3), further include
(2.3.1) draws liquid from the support grid back side, so that support grid front leaves the viscous liquid containing protein crystal；
(2.3.2) washs the support grid with 2-10%PEG solution, and the PEG solution is absorbed from the support grid back side；
(2.3.3) absorbs remaining liq with instrument, to leave protein crystal in support grid front.
In another preferred example, in step (2.1), the support grid is the support grid by hydrophilicity-imparting treatment.
In another preferred example, in step (2.1), the support grid is selected from the group: copper mesh, golden net, nickel screen or copper rhodium
Alloy network；Preferably, the support grid is copper mesh.
In another preferred example, in step (2.1), the specification of the support grid is R2/2.
In another preferred example, in step (2.1), the support grid is 400 mesh copper mesh.
In another preferred example, in step (2.2), the sample of 1~8 μ l is added in Xiang Suoshu support grid；Preferably, to the load
The sample of 2~5 μ l is added in net.
In another preferred example, after the step (2.3), repeat step (2.2), until support grid on there are
Enough protein crystals.
In another preferred example, it in step (2.4), is freezed, is freezed by the way that support grid to be inserted into liquid ethane
Sample.
In another preferred example, in the step (2.3.2), the PEG solution is the PEG solution of 1-20wt%,
The PEG solution of preferably 1-10% (w/t) (such as 5%).
In another preferred example, in the step (2.3.2), the PEG solution is PEG200 solution.
In another preferred example, in step (1), for tiny crystals protein, the processing further includes following step:
(1.1a) smashs crystal nuclear to pieces so that uniform crystal dispersion is in drop, obtains dispersion of the crystal in the liquid of pond；
Crystal is transferred to centrifuge tube together with pond liquid by (1.2a) jointly.
In another preferred example, it in step (1.1a), is smashed to pieces by needle-shaped tool.
In another preferred example, the processing further comprises the steps of:
(1.3a) is diluted the dispersion and (is diluted according to factors such as crystal size, dispersibilities, to obtain
To observable sample).
In another preferred example, in step (1), for larger crystal (micron level or several hundred microns) protein, institute
The processing stated further includes following step:
(1.1b) cuts off pipettor gun head, and protein crystal sample and crystallization solution are transferred in ep pipe；
(1.2b) carries out ultrasonic treatment to the protein crystal sample solution and/or concussion is handled；
(1.3b) precipitates big crystal, takes upper liquid as the sample solution.
In another preferred example, in step (1.2b), the ultrasonic treatment is water bath sonicator.
In another preferred example, in step (1.2b), the ultrasonic power of the ultrasonic treatment is 10%~20%.
In another preferred example, in step (1.2b), the time of the ultrasonic treatment is 0.1~1s；Preferably, it is
0.4~0.8s.
In another preferred example, in step (1.2b), the concussion processing is the concussion that is vortexed.
In another preferred example, in step (1.2b), the concussion processing is carried out 2~3 times.
In another preferred example, in step (1.3b), the big crystal of precipitating is got rid of by rotation.
In another preferred example, in step (3), the Electronic Speculum is the electron cryo-microscopy of the 120KV equipped with constituency diaphragm.
In another preferred example, in step (3), the intensity of illumination of the Electronic Speculum isIt is preferred that
Ground is
In another preferred example, in step (4), the described diffraction image collection includes: that control sample stage at the uniform velocity rotates, and
The synchronous exposure of camera is controlled, diffraction image collection is then carried out.
In another preferred example, in step (4), sample stage at the uniform velocity verts synchronous with camera exposure.
In another preferred example, the data collection use equipped with FEG filament FEI Tecnai 200Kv Electronic Speculum into
Row.
In another preferred example, the data collection, which uses, is equipped with LaB6120Kv Electronic Speculum carry out.
In another preferred example, the diffraction image is collected further include:
Measure camera response time and sample stage reaction time；
Setting starting rotation angle and termination point and sample stage rotation speed step-length and corresponding time for exposure；
Copper mesh height is adjusted, is in frozen samples always on the axis of constituency during sample stage verts；
Sample stage vert to start angle, starts data collection.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Structure (the resolution ratio for the RAC1 polypeptide sysgys that Fig. 1 is parsed using the method for the present invention)
Structure (the resolution ratio for the routine protein lysozyme polypeptide sysgys that Fig. 2 is parsed using the method for the present invention)。
Specific embodiment
The present inventor's in-depth study by long-term, develops a kind of the cold of the electron cryo-microscopy protein crystal of maturation
Freeze the preparation method of sample and the analytic method that data parse this protein structures is obtained according to Electronic Speculum.Root of the present invention
According to the own characteristic of protein nano grade tiny crystals, the screening of a whole set of crystal, electron microscopic sample preparation, data collection are had developed
And the method for structure elucidation.And ultrahigh resolution protein structure close to 1 angstrom is parsed using 120kv Electronic Speculum for the first time.
Sample treatment
It is mostly acicular crystal that amyloid aggregation core polypeptide, which is formed by tiny crystals, and is much in sea urchin
Shape, a large amount of acicular crystal aggregations are agglomerating, it is more difficult to the single crystal that processing is dispersed.Since cluster-shaped crystal is in optical microscopy
Down as it can be seen that therefore first being smashed to pieces intermediate core with needle-shaped tool, so that acicular crystal is dispersed in drop, then will
Crystal is drawn onto centrifuge tube together together with pond liquid, and is suitably diluted, and the suspension of crystal is obtained, and is used for frozen samples system
It is standby.
For bigger crystal, then need to carry out ultrasound or vortex concussion processing.Appropriate amount of sample and crystallization is molten
Liquid is transferred in the ep pipe of suitable size.Water bath sonicator: ultrasonic in the water bath sonicator for being adjusted to lowest power, time control exists
In 1s, the integrality of brilliant packet is not destroyed as far as possible.Be vortexed concussion: vortex time 5s needs multiple whirlpool if crystallite aggregation is severe
Rotation.
Frozen samples preparation
Applied sample amount, generally 4 μ l volumes are determined according to the concentration of crystal, and sample preparation is manually and Vitrobot joins
With.It the use of Quantifoil company model is generally 2/ on freezing copper mesh after appropriate amount of sample to be first loaded into hydrophilicity-imparting treatment
2,400 mesh copper mesh suck liquid with filter paper in the other side immediately after upper complete sample, are deposited in crystal on copper mesh.If crystal concentration
It is low, can multiple loading, and blotted in copper mesh reverse side.Since crystal is needed during the growth process using precipitating reagent, solution is
It is thick, cause solution that cannot be blotted completely in vitrobot, to generate thicker ice sheet on copper mesh, electronics cannot
It penetrates, the diffraction pattern or diffraction quality that cannot be got well are very poor, therefore sample preparation procedure needs as far as possible to remove solution.It is based on
Then this sucks again from the copper mesh back side, reaches we have invented being washed after sucking liquid with 4 μ l 5%PEG200 solution
Removal sample itself viscous solution and double effects antifreeze for crystal.Finally recycle Vitrobot that absorbent time is set
30s absorbs water number 2 times, and suction pressure 1 all blots surplus solution.Copper mesh is rapidly inserted into liquid ethane and is rapidly frozen,
And it is saved in liquid nitrogen environment.
Crystal screening
After the completion of sample preparation, the screening of crystallite electronic diffraction is carried out using the electron cryo-microscopy of the 120KV equipped with constituency diaphragm.Benefit
It is carried out under electron cryo-microscopy low dose mode.Wherein Search option is adjusted to LM image model, and amplification factor is 100 × left side
The right side, for determining region existing for crystal.Focus option is adjusted to diffraction mode, and camera length ratio Exposure is big by one
A step-length.The Focus option is used to determine the position size of crystal and the placement of constituency diaphragm.Exposure option is adjusted to
Diffraction mode carries out sympodium under the option and adjusts light intensity and illumination region, and adjusting option is spot size
And intensity, finally make intensity of illuminationThen light is converged into a dot using focus knob,
Determine constituency diaphragm and beam stop among optical axis simultaneously.After the completion of being adjusted under Exposure option in Search and
The parameter related with light intensity such as intensity cannot be adjusted under Focus option again.
Electronic Speculum selects the region for having diaphragm after the completion of adjusting under Search option, is switched to Focus option, is specifically chosen
The crystal of diffraction is needed, and loads the constituency diaphragm of suitable size, beam stop is placed, is switched to Exposure option, directly
Exposure, time for exposure follow sample diffraction intensity to set.
Data collection
Crystallite electron diffraction data Collection utilization software carries out.Control sample stage at the uniform velocity verts and the synchronous exposure of camera,
Camera response time and sample stage reaction time are first determined, starting rotation angle and termination point and sample are reset
Platform rotation speed step-length and corresponding time for exposure.Copper mesh height is first adjusted after the completion of setting, is made to diffraction crystal in sample stage
It is on the axis of constituency always during verting, the sample stage that then verts loads constituency diaphragm and beam to start angle
Stop, setting file store name and storage address, start data collection.
Structure elucidation
Collecting obtained data file is .mrc format, is converted into .img format text according to the camera length of measurement first
Part.The formatted file can directly using X-ray crystallography software xds carry out data processing, integrate and
Scale obtains the density .mtz file of missing phase information.Phenix software is reused, Molecular replace is and obtains
Phase information, and further refine, finally parse the structure of tiny crystals.
Main advantages of the present invention include:
A kind of structure analysis method of tiny crystals the present invention provides thickness less than 200nm, has filled up field blank.
The protein structure high resolution parsed by means of the present invention, reaches biggish crystalResolution ratio, and
It is even more to reach for protein tiny crystalsResolution ratio below.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are weight percent and weight
Number.
Embodiment 1
The structure elucidation of polypeptide SYSGYS
1) sample treatment
Intermediate core is smashed to pieces with needle-shaped tool first, acicular crystal is enable to be dispersed in drop, it then will be brilliant
Body is drawn onto centrifuge tube together together with pond liquid, and is suitably diluted, and the suspension of crystal is obtained, and is prepared for frozen samples.
2) prepared by frozen samples
Applied sample amount is determined according to the concentration of crystal, and volume is 4 μ l, and sample preparation is manually and Vitrobot is combined.First
It the use of Quantifoil company model is generally 2/2,400 on freezing copper mesh after appropriate amount of sample to be loaded into hydrophilicity-imparting treatment
Mesh copper mesh sucks liquid with filter paper in the other side immediately after upper complete sample, is deposited in crystal on copper mesh.It, can if crystal concentration is low
Multiple loading, and blotted in copper mesh reverse side.It is washed after sucking liquid with 4 μ l 5%PEG200 solution, then again from copper
The net back side sucks, and has reached removal sample itself viscous solution and the double effects antifreeze for crystal.Finally recycle
Absorbent time 30s is arranged in Vitrobot, absorbs water number 2 times, suction pressure 1 all blots surplus solution.Copper mesh is rapidly inserted into
It is rapidly frozen in liquid ethane, and is saved in liquid nitrogen environment.
3) crystal screens
After the completion of sample preparation, the screening of crystallite electronic diffraction is carried out using the electron cryo-microscopy of the 120KV equipped with constituency diaphragm.Benefit
It is carried out under electron cryo-microscopy low dose mode.Wherein Search option is adjusted to LM image model, and amplification factor is 100 × left side
The right side, for determining region existing for crystal.Focus option is adjusted to diffraction mode, and camera length ratio Exposure is big by one
A step-length.The Focus option is used to determine the position size of crystal and the placement of constituency diaphragm.Exposure option is adjusted to
Diffraction mode carries out sympodium under the option and adjusts light intensity and illumination region, and adjusting option is spot size
And intensity, finally make intensity of illuminationThen light is converged into a dot using focus knob,
Determine constituency diaphragm and beam stop among optical axis simultaneously.After the completion of being adjusted under Exposure option in Search and
The parameter related with light intensity such as intensity cannot be adjusted under Focus option again.
Electronic Speculum selects the region for having diaphragm after the completion of adjusting under Search option, is switched to Focus option, is specifically chosen
The crystal of diffraction is needed, and loads the constituency diaphragm of suitable size, beam stop is placed, is switched to Exposure option, directly
Exposure, time for exposure follow sample diffraction intensity to set.
4) data collection
Crystallite electron diffraction data Collection utilization software carries out.Control sample stage at the uniform velocity verts and the synchronous exposure of camera,
Camera response time and sample stage reaction time are first determined, starting rotation angle and termination point and sample are reset
Platform rotation speed step-length and corresponding time for exposure.Copper mesh height is first adjusted after the completion of setting, is made to diffraction crystal in sample stage
It is on the axis of constituency always during verting, the sample stage that then verts loads constituency diaphragm and beam to start angle
Stop, setting file store name and storage address, start data collection.
5) structure elucidation
Collecting obtained data file is .mrc format, is converted into .img format text according to the camera length of measurement first
Part.The formatted file can directly using X-ray crystallography software xds carry out data processing, integrate and
Scale obtains the density .mtz file of missing phase information.Phenix software is reused, Molecular replace is and obtains
Phase information, and further refine, finally parse the structure of polypeptide SYSGYS.
Parsing result is as shown in Figure 1, the resolution ratio of the SYSGYS polypeptide structure parsed reaches
Embodiment 2
The parsing of the structure of lysozyme conventional crystalline.
1) sample treatment
Appropriate amount of sample and crystallization solution are transferred in the ep pipe of suitable size.Water bath sonicator: it is being adjusted to lowest power
Water bath sonicator in ultrasound, ultrasonic power 20%, water bath time 0.5s do not destroy the integrality of brilliant packet as far as possible.Sample after ultrasound
Sample is got rid of in rotation, is made bulk deposition in test tube bottom, is beaten easily upper sample sample preparation.
Frozen samples preparation, crystal screening, data collection and structure elucidation step are the same as embodiment 1.
Parsing result is as shown in Fig. 2, the resolution ratio of the structure for the lysozyme conventional crystalline being precipitated is
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (10)
1. a kind of analytic method of protein tiny crystals structure, which is characterized in that include the following steps:
(1) protein tiny crystals sample is handled, obtains sample solution, the sample solution is that the protein containing dispersion is brilliant
The solution of body；
(2) preparation of frozen samples: freezing processing is carried out to the sample solution, obtains frozen samples；
(3) crystal screens: finding the crystal for being used for analytic structure by Electronic Speculum, and determines the parsing item of crystal positions and Electronic Speculum
Part；
(4) data collection: the diffraction image of crystal different angle is collected；
(5) structure elucidation: according to the diffraction image of different angle, parsing obtains the structure of protein crystal.
2. analytic method as described in claim 1, which is characterized in that in step (2), further comprise the steps of:
(2.1) support grid is provided；
(2.2) the support grid surface of Xiang Suoshu adds sample liquid (preferably 2-5 μ l)；
(2.3) liquid is removed, falls to protein crystal in the support grid；
(2.4) protein crystal of the support grid and surface is rapidly frozen, obtains frozen samples.
3. analytic method as claimed in claim 2, which is characterized in that in step (2.3), further include
(2.3.1) draws liquid from the support grid back side, so that support grid front leaves the viscous liquid containing protein crystal；
(2.3.2) washs the support grid with 2-10%PEG solution, and the PEG solution is absorbed from the support grid back side；
(2.3.3) absorbs remaining liq with instrument, to leave protein crystal in support grid front.
4. analytic method as claimed in claim 2, which is characterized in that in step (2.1), the support grid is selected from the group: copper
Net, golden net, nickel screen or copper rhodium alloy net；Preferably, the support grid is copper mesh.
5. the method as described in claim 1, which is characterized in that in step (1), for tiny crystals protein, the place
Reason further includes following step:
(1.1a) smashs crystal nuclear to pieces so that uniform crystal dispersion is in drop, obtains dispersion of the crystal in the liquid of pond；
Crystal is transferred to centrifuge tube together with pond liquid by (1.2a) jointly.
6. the method as described in claim 1, which is characterized in that in step (1), for larger crystal, (micron level is several
Hundred microns) protein, the processing further includes following step:
(1.1b) cuts off pipettor gun head, and protein crystal sample and crystallization solution are transferred in ep pipe；
(1.2b) carries out ultrasonic treatment to the protein crystal sample solution and/or concussion is handled；
(1.3b) precipitates big crystal, takes upper liquid as the sample solution.
7. the method as described in claim 1, which is characterized in that in step (3), the Electronic Speculum is equipped with constituency diaphragm
The electron cryo-microscopy of 120KV.
8. the method as described in claim 1, which is characterized in that in step (3), the intensity of illumination of the Electronic Speculum isPreferably, it is
9. the method as described in claim 1, which is characterized in that in step (4), the diffraction image collection includes: control
Sample stage at the uniform velocity rotates, and controls the synchronous exposure of camera, then carries out diffraction image collection.
10. method as claimed in claim 9, which is characterized in that in step (4), the diffraction image is collected further include:
Measure camera response time and sample stage reaction time；
Setting starting rotation angle and termination point and sample stage rotation speed step-length and corresponding time for exposure；
Copper mesh height is adjusted, is in frozen samples always on the axis of constituency during sample stage verts；
Sample stage vert to start angle, starts data collection.
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