New methods for high-throughput cryo-em data collection

Abstract

The application provides a high-throughput cryo-EM data collection method: after the sample stage is moved to a target position, the sample stage is moved reversely twice, the stabilization time of the sample stage is shortened, and the collection speed of the cryo-EM is improved. The detailed steps of the optimized sample stage movement are as follows: the sample stage is moved to a position required for shooting in a conventional manner; the sample stage is moved reversely by 0.1 mu m for the first time, the sample stage is moved reversely by 0.02-0.08 mu m on the basis of the previous step, and then is stabilized for 2 seconds.

Description

Technical Field

[0001] This invention relates to a novel method for high-throughput cryo-electron microscopy data collection. Background Technology

[0002] Cryo-electron microscopy (cryo-EM) single-particle 3D reconstruction is a crucial technique for obtaining high-resolution 3D structures of biomolecules. This technique requires acquiring a sufficient number of high-quality images of the same homogeneous sample using cryo-EM. However, the current limited cryo-EM timeframe is increasingly at odds with the growing demands of users. Therefore, continuously improving cryo-EM data acquisition speed and data quality are two core issues that most cryo-EM platforms are focusing on.

[0003] During cryo-electron microscopy (cryo-EM) data acquisition, the imaging target needs to be repeatedly moved to the beam center for imaging. Traditionally, this is accomplished by moving the stage of the electron microscope. The stage movement is achieved through a mechanical gear transmission process, so after the stage moves to the target position, the gears experience tension, causing the stage to drift. To obtain high-quality data, the stage needs to stabilize for a period of time after movement before image acquisition to release the tension and improve image quality. This stabilization time is typically 8-20 seconds, accounting for about 30-40% of the data acquisition time, which is a limiting step in data acquisition speed. Using a Titan Krios G2 (Thermo Fisher Scientific) equipped with a Gatan K2 camera, this method allows for a cryo-EM acquisition speed of approximately 60 images per hour.

[0004] Besides moving the sample stage, beam spot translation can also be used to acquire images of the target. The principle of beam spot translation is to change the current in the magnetic lens coil to deflect the electron beam to the target area, thus imaging the target region. Beam spot translation can save time on sample stage movement and stabilization, increasing data collection speed. However, beam spot translation introduces additional aberrations into the collected data, which, without additional correction, can lead to a decrease in the resolution of the 3D reconstruction.

[0005] The main drawbacks of existing technologies are: the method of moving the sample stage takes a long time to stabilize and the data collection speed is slow; the method of beam spot translation has a fast data collection speed, but it introduces additional aberrations. Summary of the Invention

[0006] To address the technical problems in the background art, this application provides a novel method for high-throughput cryo-electron microscopy data collection, which shortens the stabilization time of the sample stage by moving it to the target position twice in the opposite direction, thereby improving the collection speed of cryo-electron microscopy.

[0007] The objective of this application is achieved through the following technical solution:

[0008] A novel method for high-throughput cryo-electron microscopy data collection includes the following steps:

[0009] 1) Preparation of frozen samples

[0010] 2) Data collection

[0011] Data was collected using cryo-electron microscopy in conjunction with automated collection software;

[0012] 3) Sample stage movement method

[0013] The movement of the sample stage is controlled by the automatic collection software. The movement pattern is as follows: after normal movement, move in the opposite direction by 0.1 μm, then move in the opposite direction by 0.02-0.08 μm, and then stabilize for 2 seconds.

[0014] 4) Use software to correct the drift of the collected data.

[0015] Step 1 is as follows:

[0016] 1.1) Network Preparation

[0017] The carrier screen is an Au 1.2 / 1.3 300 mesh screen, which is then cleaned using a plasma cleaner.

[0018] 1.2) Protein Sample Preparation

[0019] The purified membrane protein LolCDE complex was used as the test sample, with a protein concentration of 2 mg / ml.

[0020] 1.3) Sample Preparation

[0021] 3 μL of protein was dropped onto the treated grid and sample was prepared using Vitrobot. The preparation conditions were: temperature 20°C, relative humidity 100%, blot force 0, and blot time 3 s.

[0022] The relevant parameters for data collection in step 2 are set as follows: nominal magnification is 29000x, exposure time is 6.4s, 5 frames per second, and total electron dose is...

[0023] Step 3 is preferably performed by moving the sample stage in an automatic collection software manner, which involves moving it in the opposite direction for 0.1 μm after normal movement, then moving it in the opposite direction for 0.02 μm, and then stabilizing it for 2 seconds.

[0024] Step 4 is preferably performed by using Motioncor2 software to correct the drift of the collected data.

[0025] The advantage of this application is that after the sample stage is moved to the target position, the time for the sample stage to stabilize is shortened by moving the sample stage in reverse twice, thereby improving the collection speed of cryo-electron microscopy. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the movement of the sample stage according to the present invention;

[0027] Figure 2 This is a graph showing the correlation analysis results between the average image drift rate and the sample stage movement mode of the present invention. Detailed Implementation

[0028] The preferred embodiments of this application will now be described in detail with reference to the accompanying drawings. To enable those skilled in the art to better understand the solutions of this application, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the manufacturer.

[0029] Example 1

[0030] 1. Preparation of frozen samples

[0031] (1) Network preparation

[0032] The carrier screen was a Quantifoil (Au 1.2 / 1.3 300 mesh) and was then plasma cleaned using a plasma cleaner (PELCO, Easyglow) (20mA, 120s).

[0033] (2) Protein sample preparation

[0034] The purified membrane protein LolCDE complex was used as the test sample, with a protein concentration of 2 mg / ml.

[0035] (3) Sample preparation

[0036] 3 μL of protein was dropped onto the treated grid and sample was prepared using Vitrobot (Thermo Fisher). The preparation conditions were: temperature 20°C, relative humidity 100%, blot force 0, and blot time 3 s.

[0037] 2. Data Collection

[0038] Data collection was performed using a 300kV cryo-electron microscope (Titan Krios G2, Thermo Fisher) equipped with a K2 camera (Gatan). SerialEM (3.8) automatic data collection software was used, with the following parameters set: nominal magnification 29000x, exposure time 6.4s, 5 frames per second, and total electron dose...

[0039] 3. Sample stage movement method

[0040] The sample stage movement is controlled by the automatic collection software SerialEM. After a normal movement, it moves in the opposite direction by 0.1 μm, then in the opposite direction by 0.02 μm, and then stabilizes for 2 seconds.

[0041] 4. Data Analysis

[0042] The collected data were drift-corrected and analyzed using Motioncor2 software.

[0043] Example 2

[0044] The sample stage movement was controlled by the automatic collection software SerialEM. The movement pattern was as follows: normal movement followed by a reverse movement of 0.1 μm, then a reverse movement of 0.04 μm, and then stabilization for 2 seconds. The rest was the same as in Example 1.

[0045] Example 3

[0046] The sample stage movement was controlled by the automatic collection software SerialEM. The movement pattern was as follows: normal movement followed by a reverse movement of 0.1 μm, then a reverse movement of 0.08 μm, and then stabilization for 2 seconds. The rest was the same as in Example 1.

[0047] Comparative Example 1

[0048] The movement of the sample stage is controlled by the automatic collection software SerialEM. The movement mode is: normal movement followed by 2 seconds of stabilization.

[0049] The rest is the same as in Example 1.

[0050] Comparative Example 2

[0051] The movement of the sample stage is controlled by the automatic collection software SerialEM. The movement mode is: normal movement followed by stabilization for 16 seconds.

[0052] The rest is the same as in Example 1.

[0053] like Figure 2The correlation analysis results of the average drift rate and the sample stage movement method are shown in the figure. The bars in the figure represent the drift rate results after the sample stage moves normally without introducing additional reverse movement and stabilizes for 2 s; after normal movement, reverse movement of 0.1 μm, then reverse movement of 0.02 μm, and then stabilizes for 2 s; after normal movement, reverse movement of 0.1 μm, then reverse movement of 0.04 μm, and then stabilizes for 2 s; after normal movement, reverse movement of 0.1 μm, then reverse movement of 0.08 μm, and then stabilizes for 2 s; and after normal movement without introducing additional reverse movement and stabilizing for 16 s.

[0054] Depend on Figure 2 It can be concluded that by introducing two additional reverse movements, the first reverse movement being 0.1 μm and the second reverse movement being 0.02-0.08 μm, the time required for the sample stage to stabilize is reduced from 16 s to 2 s, and the drift rate is slightly better than that of stabilization alone in 16 s, thereby improving the data collection speed of cryo-electron microscopy.

[0055] Any embodiment of this application can be used as an independent technical solution or combined with other embodiments. All patents and publications mentioned in this application represent publicly available technologies that this application can use. All patents and publications cited herein are also listed in the references as if each publication were individually referenced. This application can be implemented in the absence of any one or more elements, or one or more limitations, without any particular limitation stated herein. The terminology and expressions used herein are descriptive methods and are not intended to be limiting, nor is there any intention to exclude any equivalent features from the terms and interpretations described herein; however, it is understood that any suitable changes or modifications can be made within the scope of this application and the claims. It is understood that the embodiments described in this application are examples of embodiments and features in some examples, and any person skilled in the art can make changes and variations based on the essence of the description in this application. These changes and variations are also considered to fall within the scope of this application and the limitations of the independent and appended claims.

Claims

1. A novel method for high-throughput cryo-electron microscopy data collection, characterized in that, Includes the following steps: 1) Preparation of frozen samples 2) Data collection Data was collected using cryo-electron microscopy in conjunction with automated collection software; 3) Sample stage movement method The movement of the sample stage is controlled by the automatic collection software. The movement pattern is as follows: after normal movement, move in the opposite direction by 0.1 μm, then move in the opposite direction by 0.02-0.08 μm, and then stabilize for 2 seconds. 4) Use software to correct the drift of the collected data and calculate the offset; Step 1 is as follows: 1.1) Network Preparation The carrier screen is an Au 1.2 / 1.3 300 mesh screen, which is then cleaned using a plasma cleaner. 1.2) Protein Sample Preparation The purified membrane protein LolCDE complex was used as the test sample, with a protein concentration of 2 mg / ml. 1.3) Sample Preparation 3 μL of protein was dropped onto the treated grid and sample was prepared using Vitrobot. The preparation conditions were: temperature 20°C, relative humidity 100%, blot force 0, and blot time 3 s.

2. The novel method for high-throughput cryo-electron microscopy data collection according to claim 1, characterized in that, The relevant parameters for data collection in step 2 are set as follows: nominal magnification is 29,000x, exposure time is 6.4s, 5 frames per second, and total electron dose is 50e- / Å2.

3. The novel method for high-throughput cryo-electron microscopy data collection according to claim 1, characterized in that, Step 3 is as follows: The movement of the sample stage is controlled by the automatic collection software. The movement method is: after normal movement, move in the opposite direction by 0.1 μm, then move in the opposite direction by 0.02 μm, and then stabilize for 2 seconds.

4. The novel method for high-throughput cryo-electron microscopy data collection according to claim 1, characterized in that, Step 4 is to use Motioncor2 software to perform drift correction on the collected data.

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