CN113325019B - Method for preparing sample of phycobilisome of blue algae by using cryoelectron microscope - Google Patents
Method for preparing sample of phycobilisome of blue algae by using cryoelectron microscope Download PDFInfo
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Abstract
The invention discloses a blue algae phycobilisome sample preparation method by a cryoelectron microscope, which overcomes three difficulties of depolymerization, poor contrast and dominant orientation of the conventional cryoelectron sample preparation of phycobilisome by crosslinking blue algae phycobilisome protein purified in vitro and adopting a sample loading mode of 'loading the sample on the front side and diluting the sample on the back side', can analyze the near-atomic-level high-resolution structure information of the blue algae phycobilisome, and provides an effective method for analyzing the high-resolution structure of various blue algae phycobilisome by using the cryoelectron microscope in the future.
Description
Technical Field
The invention relates to the field of preparation of a cryoelectron microscope sample of a biological sample, in particular to a method for preparing a blue algae phycobilisome cryoelectron microscope sample.
Background
The cyanobacteria phycobilisome is a large-scale light-capturing antenna protein complex which is positioned on one side of the outside of an inner vesicle membrane of a cyanobacteria cell, which faces to a substrate, and can absorb light energy and transmit the energy to a photosystem reaction center. At present, many biochemical experiments aiming at phycobilisomes are used for researching the assembly and energy transfer mechanisms of the phycobilisomes, but many questions are still unclear, so that the analysis of high-resolution structural information of the phycobilisomes is urgently needed, and the assembly and energy transfer mechanisms of the phycobilisomes are understood by combining the biochemical results.
Phycobilisomes of cyanobacteria have been purified in vitro in the last 70 th century, but the near-atomic-scale high-resolution structures of phycobilisomes of cyanobacteria have not been resolved for half a century so far. The phycobilisome purified in vitro can exist stably only in an environment with high phosphate (0.6-1.0 mol/L) and normal temperature (16-28 ℃), and the crystal does not grow in the environment, so the structure cannot be analyzed by the commonly used X-ray crystal diffraction (X-ray), the molecular weight of the phycobilisome of the blue algae is about 6000kDa, the structure cannot be analyzed by nuclear magnetic resonance, and finally the structure is analyzed by the cryoelectron microscope technology.
However, the analysis of cyanobacterial phycobilisome structure by cryoelectron microscopy (Cryo-EM) has three difficulties so far: 1. the cyanobacteria phycobilisome sample only exists stably in a normal temperature environment, and the sample is quickly frozen in liquid ethane at the temperature of about-150 ℃ in the process of preparing the sample by a cryoelectron microscope, so the cyanobacteria phycobilisome sample is usually depolymerized through the sample preparation by the cryoelectron microscope; 2. the cyanobacteria phycobilisome sample can exist stably only in a high phosphate (0.6-1.0 mol/L) environment, and although Cryo-EM can image under high-concentration phosphate for maintaining the stability of the compound, the contrast of the sample in the environment under a cryoelectron microscope is reduced rapidly, so that the resolution of the resolved protein complex structure is not high; 3. because the blue algae phycobilisome is usually in a disc shape, and the part of the bottom of the blue algae phycobilisome connected with the thylakoid membrane is provided with a hydrophobic structure domain, the conventional freezing sample preparation of the blue algae phycobilisome still has dominant orientation, which can greatly influence the correct establishment of a three-dimensional reconstruction model thereof.
Disclosure of Invention
The invention aims to provide a sample preparation method of a cyanobacteria phycobilisome cryoelectron microscope, which overcomes three difficulties of depolymerization, poor contrast and dominant orientation of the conventional cryosample preparation of the phycobilisome and provides an effective method for analyzing high-fraction structures of various cyanobacteria phycobilisome by using the cryoelectron microscope.
In order to achieve the purpose, the invention solves the problem of low-temperature depolymerization of the phycobilisome by crosslinking 1, 5-Glutaraldehyde (GA), then rapidly reduces the concentration of phosphoric acid in the sample by using a method of 'front sample loading and reverse rapid dilution' to solve the problem of contrast, and adds a detergent such as NP40 into a dilution buffer solution to solve the problem of dominant orientation of the sample, so that the freeze sample preparation of the phycobilisome can obtain a sample with high integrity, good contrast and no dominant orientation, and an effective means is provided for the subsequent single-particle sample data acquisition and analysis so as to analyze the high-resolution structure of the phycobilisome. Specifically, the technical scheme of the invention is as follows:
a method for preparing a sample of phycobilisomes of cyanobacteria by a cryoelectron microscope comprises the following steps:
(1) purifying blue algae phycobilisomes in vitro by sucrose gradient centrifugation;
(2) removing sucrose in the blue algae phycobilisome solution purified in vitro, concentrating to obtain phycobilisome protein solution with the concentration of 9-12 mg/mL, adding 1, 5-Glutaraldehyde (GA) with the final concentration of 0.005-0.015% (w/v) for crosslinking for 1-2 minutes, and adding Tris-HCl buffer solution with the pH value of 7.0 to terminate the reaction;
(3) adopting a method of 'front sample loading and back dilution' to prepare a sample by a cryoelectron microscope, comprising the following steps:
(3-1) carrying out hydrophilization treatment on the two sides of the mesh for freezing sample preparation;
(3-2) loading the crosslinked phycobilisome protein sample on the front surface of the mesh subjected to hydrophilization treatment;
(3-3) quickly adding a pH7.0Tris-HCl buffer solution containing a nonionic detergent and having the same volume as the sample loading protein sample into the reverse side of the grid, and blowing and sucking for 2-3 times;
(3-4) repeating step (3-3) to rapidly reduce the salt concentration of the loaded protein sample to 1/3.
The method for purifying the cyanobacteria phycobilisome in vitro in the step (1) is a method known in the prior art.
In the step (2), the blue algae phycobilisome purified in vitro is washed by a sugar-free buffer solution (usually a sodium phosphate and/or potassium phosphate buffer solution), sucrose is removed, and then the blue algae phycobilisome is concentrated into a storage solution with the phycobilisome protein concentration of 9-12 mg/mL for later use, and the phycobilisome protein storage solution is taken and added with a cross-linking agent for cross-linking before the preparation of a frozen electron microscope.
In the step (3), the sample preparation operation of other phycobilisomes which are not particularly described is the same as the sample preparation operation of a conventional cryoelectron microscope.
Preferably, in the step (2), the final concentration of the added Tris-HCl buffer solution with the pH of 7.0 is 10-50 mu M.
Preferably, the loading operation is performed in the step (3) at 18 ℃ and 100% humidity.
In the step (3-1), the grid is preferably a gold grid, and the gold grid can be subjected to hydrophilization treatment on both sides by a glow discharge method.
In the step (3-3), the non-ionic detergent may be selected from one of NP40 (ethylphenylpolyethylene glycol), DDM (dodecyl-beta-D-maltoside), and the like, the pH7.0Tris-HCl buffer is preferably 10-50 mM pH7.0Tris-HCl buffer, and the content of the non-ionic detergent is preferably 0.01-0.05% (w/v).
Generally, the sample loading volume of the crosslinked phycobilisome protein sample in the step (3-2) is 3-4 muL, and the salt concentration (phosphate) of the sample loading protein sample is quickly reduced to 1/3 through blowing and sucking of the equal volume of buffer solution twice in the steps (3-3) and (3-4).
The blue algae phycobilisome cryoelectron microscope sample preparation method overcomes three difficulties of depolymerization, poor contrast and dominant orientation of the conventional phycobilisome cryoelectron microscope sample preparation by crosslinking phycobilisome protein and adopting a sample loading mode of 'loading the sample on the front side and diluting the sample on the back side', can analyze the near-atomic-level high-resolution structure information of the blue algae phycobilisome, and provides an effective method for analyzing the high-resolution structure of various blue algae phycobilisome by using a cryoelectron microscope in the future.
Drawings
FIG. 1 is a gradient centrifugation chart of sucrose from phycobilisome purified cyanobacteria in vitro, and the arrows indicate the bands of intact phycobilisome.
FIG. 2 is a schematic diagram of the operation of the "loading on front side and dilution on back side" steps described in the method of the present invention.
FIG. 3 shows the effect of freezing the prepared sample and the resolution of the analytical structure using the prepared sample data, wherein: a shows that the phycobilisome is in a five-core eight-rod disc shape through transmission electron microscope observation after negative staining; b is a blue algae phycobilisome sample prepared by the method is observed under a 300kv cryoelectron microscope; c is a three-dimensional average reconstructed picture after data are collected by utilizing single particle analysis; d is the overall resolution chart of the resolved cyanobacteria phycobilisomes, which is resolved to the resolution of near atomic level.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the following examples,% is by mass unless otherwise specified.
Blue bacterium Anabaena sp.PCC 7120 (derived from fresh water algae seed bank (FACHB) of Chinese academy of sciences) is grown in BG11 liquid to illuminate fluorescent lamp light source with light intensity of 25uE/m2S, temperature 30 ℃ and introduction of 1% CO2(v/v) air.
Wherein the Trace metal mix comprises the following components:
example 1 in vitro purification of cyanobacteria phycobilisomes
1. Buffer A (0.9M sodium phosphate Buffer pH 8.0, 1L containing 300.41g Na2HPO4·12H2O，9.55g NaH2PO4·2H2O and 10mM EDTA) and Buffer B (0.75M potassium phosphate Buffer pH 8.0, 1 liter 160.9g K2HPO4，6.12g KH2PO4And 10mM EDTA), Buffer A and Buffer B are used as storage solutions, and when in use, the Buffer A and the Buffer B are mixed in equal volume to obtain Buffer C;
2. sterilizing solid sucrose, and preparing 1M, 0.85M, 0.70M, 0.55M, 0.40M and 0.25M sucrose solutions by using Buffer C;
3. collecting 2L cyanobacteria Anabaena sp.PCC 7120 bacterial liquid, centrifuging at 8000rpm, collecting precipitate, grinding and crushing by liquid nitrogen, and keeping out of the sun as much as possible in the following steps;
4. suspending the ground blue algae powder with 25mL Buffer C (PMSF (phenylmethylsulfonyl fluoride), benzamidine and 6-aminocaproic acid, all of which have a final concentration of 0.1mM, are added in advance), and centrifuging at 4 ℃ and 3000rpm for 10min to obtain a supernatant;
5. adding Triton X-100 to a final solubility of 2% (v/v), shaking gently in a shaking table for 30min, centrifuging at 20000rpm for 30min, collecting middle layer blue liquid, and removing upper and lower layer impurities;
6. preparing 1M, 0.85M, 0.70M, 0.55M, 0.40M and 0.25M sucrose solutions by using Buffer C, adding the sucrose solutions into an ultracentrifuge centrifuge tube from low concentration to high concentration in a ratio of 3:3:3:4:4:5, adding the prepared blue phycobilisome crude extract accounting for 10 percent of the total volume of the centrifuge tube, and ultracentrifuging at 35000rpm overnight;
7. a dark blue strip at the interface of 0.70M sucrose is absorbed by a needle with a hook to obtain a complete phycobilisome (see figure 1), and the shape of the phycobilisome is a five-core eight-rod disc shape as shown in figure 3 a through transmission electron microscope observation after negative staining.
Example 2 preparation of cryoelectron microscope sample of cyanobacteria phycobilisomes
(1) Blue algae phycobilisome desugarization concentration cross-linking
1. The phycobilisomes from cyanobacteria purified in vitro were concentrated using Millipore 30kDa ultrafiltration tubes, washed simultaneously with sugar-free buffer C to remove sucrose, and finally concentrated to a 10mg/mL stock solution of phycobilisomes proteins.
2. The reaction was stopped by addition of 1, 5-Glutaraldehyde (GA) at a final concentration of 0.01% (w/v) for crosslinking for 2 minutes, followed by addition of Tris-HCl at a final concentration of 50. mu.M pH 7.0. The phycobilisome sample after cross-linking treatment is used for subsequent protein cryoelectron microscope sample preparation.
(2) Freezing sample preparation of blue algae phycobilisome
1. The gold mesh was subjected to hydrophilization treatment by glow discharge on both sides with 300 mesh gold mesh of R1.2/1.3 available from Quantifol.
2. And (3) carrying out preparation of a frozen electron microscope sample on the crosslinked protein by using a FEI Vitrobot frozen electron microscope sample preparation instrument under the conditions of 18 ℃ and 100% humidity. Except adopting a method of 'front sample loading and reverse side rapid dilution', other operations are conventional protein sample cryo-electron microscope conventional operations.
(3) "front loading, back fast dilution" (shown in FIG. 2) procedure was as follows:
the method comprises the following steps: and (3.5) mu L of the blue algae phycobilisome sample after crosslinking is loaded on the front surface of the gold mesh and is adsorbed for 60 seconds.
Step two: 3.5 μ L of 50mM Tris-HCl pH7.0 buffer containing 0.05% (w/v) NP40 was added rapidly to the reverse side of the gold mesh, and aspiration was performed 2-3 times.
Step three: repeating the step two more times, and finally quickly reducing the phosphate concentration of the phycobilisomes to 1/3.
Example 3 cryoelectron microscopy sample data collection for cyanobacteria phycobilisomes and analysis of their structures by single particle analysis
As shown in b in FIG. 3, when the cyanobacteria phycobilisome sample prepared by the method of the present invention is observed under a 300kv cryoelectron microscope, the contrast and integrity are both ideal, and no dominant orientation is present.
After a good sample of phycobilisomes is screened by a cryoelectron microscope, data are collected by a 300kv cryoelectron microscope. In fig. 3, c is a three-dimensional average reconstructed picture after data collection by single particle analysis. D in fig. 3 is a resolved blue algae phycobilisome overall resolution map, which has been resolved to a near atomic resolution.
Claims (10)
1. A method for preparing a sample of phycobilisomes of cyanobacteria by a cryoelectron microscope comprises the following steps:
1) purifying blue algae phycobilisomes in vitro by sucrose gradient centrifugation;
2) removing sucrose in the blue algae phycobilisome solution purified in vitro, concentrating to obtain phycobilisome protein solution with the concentration of 9-12 mg/mL, adding 1, 5-glutaraldehyde with the final concentration of 0.005-0.015% (w/v) for crosslinking for 1-2 minutes, and adding Tris-HCl buffer solution with the pH of 7.0 to terminate the reaction;
3) adopting a method of 'front sample loading and back dilution' to prepare a sample by a cryoelectron microscope, comprising the following steps:
3-1) carrying out hydrophilization treatment on two sides of a net for freezing sample preparation, wherein the net is a gold net;
3-2) loading the crosslinked phycobilisome protein sample on the front surface of the carrier net after hydrophilization treatment;
3-3) quickly adding a Tris-HCl buffer solution with the pH value of 7.0 and containing a nonionic detergent, which is equal to the volume of the loaded protein sample, into the reverse side of the carrier net, and blowing and sucking for 2-3 times;
3-4) repeating step 3-3) to rapidly reduce the salt concentration of the loaded protein sample to 1/3.
2. The method as claimed in claim 1, wherein the blue algae phycobilisome purified in vitro is washed with sugar-free buffer solution in step 2) to remove sucrose, and then concentrated to a solution with phycobilisome protein concentration of 9-12 mg/mL.
3. The method according to claim 2, wherein the sugar-free buffer used in step 2) is a sodium phosphate and/or potassium phosphate buffer.
4. The method of claim 1, wherein the final concentration of the Tris-HCl buffer solution added in step 2) at pH7.0 is 10-50 μ Μ.
5. The method of claim 1, wherein step 3) is performed at 18 ℃ and 100% humidity.
6. The method of claim 1, wherein the gold mesh is hydrophilized on both sides in step 3-1) by means of glow discharge.
7. The method according to claim 1, wherein the loading volume of the crosslinked phycobilisome protein sample in step 3-2) is 3 to 4 μ L.
8. The method of claim 1, wherein the non-ionic detergent in step 3-3) is NP40 or dodecyl-beta-D-maltoside.
9. The method of claim 1, wherein the Tris-HCl buffer solution of pH7.0 in step 3-3) is 10-50 mM of pH7.0Tris-HCl buffer solution, and the content of the nonionic detergent is 0.01-0.05% (w/v).
10. The method of claim 1, wherein the cyanobacterium is Anabaena sp.
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| CN1524880A (en) * | 2003-02-28 | 2004-09-01 | 中国科学院过程工程研究所 | Protein cross linking agent and method therefor |
| CN109001003A (en) * | 2018-06-22 | 2018-12-14 | 中国科学院长春应用化学研究所 | For freezing the preparation method of electron microscope observation cell membrane sample |
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| CN1524880A (en) * | 2003-02-28 | 2004-09-01 | 中国科学院过程工程研究所 | Protein cross linking agent and method therefor |
| CN109001003A (en) * | 2018-06-22 | 2018-12-14 | 中国科学院长春应用化学研究所 | For freezing the preparation method of electron microscope observation cell membrane sample |
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