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11:52 min
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May 29th, 2021
DOI :
May 29th, 2021
•0:04
Introduction
0:47
Performing an Inventory and Screening for Ice and Sample Quality
4:07
Single Particle CryoEM Data Collection
9:44
Results: Single Particle Cryo-Electron Microscopy: Screening for 3D Classification and Refinement
11:04
Conclusion
필기록
Single-particle cryo-EM analysis has become a routine technique in the structural biologist's toolbox applicable to a wide range of specimens including membrane proteins, amyloid fibrils, nucleic acid binding proteins and viruses. Once sample preparation has been completed and the grids loaded into the microscope, data collection can be carried out remotely in many cryo-EM facilities such as the Astbury Biostructure Laboratory and eBIC. Major barriers to successful structure determination often lie in the sample preparation and grid screening stages with multiple iterations sometimes required through this process.
Here, we will demonstrate remote grid screening and single-particle data acquisition. In the auto-loader tab of the microscope user interface, tab out the options dialog using the arrow and press the inventory button. This will sequentially check each position in the cassette to determine if a cartridge is present.
The inventory will run on each slot sequentially. When all occupied slots are mapped, the inventory will stop. Highlight the grid to be transferred to the microscope column and click load.
The slot label will turn from blue to yellow once the grid has been successfully loaded onto the stage. To screen the grids, open EPU software. On the preparation page, select acquisition optics and settings, then select the atlas preset from the dropdown menu.
Choose appropriate beam setting presets and press set to push the parameters to the microscope. Press to open column valves and insert the flu screen. Check that a beam is visible and sufficiently spread and centered to cover the detector.
If necessary, navigate to a thinner region of the grid using the joystick or virtual hand panels to control stage movements in X and Y.Lift the flu screen and take an image using the preview button in EPU. In EPU, go to the atlas page and press new session. Select the MRC image format and enter a suitable folder name and location for saving the screening session, then click apply.
Select screening from the menu on the left. Tick the checkboxes next to each grid to have an atlas montage acquired, then start the screening session in EPU. An atlas is acquired for each checked grid and available grid squares are listed upon completion.
Each atlas can be viewed by highlighting it on the screening page. When finished, review the collected atlases and identify the grids suitable for assessing sample quality at higher magnifications. Highlight the chosen grid on the EPU screening menu and click load sample.
From the atlas screening menu, select the grid presently loaded and move the stage to a grid square containing the filled holes by right-clicking over the desired location on the grid image and selecting move stage here. Return to EPU preparation page and select the grid square preset. Open the EPU auto-functions page and run auto-eucentric by stage tilt with the grid square preset to move the sample to eucentric height.
In EPU preparation, take a new grid square preview image. Note the gray values across different holes indicating differing ice thicknesses. Move the stage over a hole using right-click and move stage here.
Select the hole or eucentric height preset, then click preview. Select data acquisition preset and set a magnification that allows easy identification of the particles. Set the defocus offset to negative three to five micrometers.
Iterate through steps to reassess a range of ice thickness for particle distribution, orientation, and contamination across the grid. Depending on microscope availability, continue directly to data collection or grids can be removed from the microscope for future collection including at other sites. Collect an atlas if one is not available from screening by setting up atlas settings and selecting grid to acquire atlas.
Acquire atlas and click on grid location to see the output. Once the atlas is complete, define each of the beam setting presets according to the experimental needs of the project. Perform image shift calibrations.
Set up the EPU session by selecting the EPU page session setup and then selecting either new session or new from preferences. After selecting new session, a popup will appear providing an option to use previous settings. The settings will automatically load from the previous to the current EPU session.
Alternatively, select new from preferences and pick a file with saved preferences to preload this information into the current EPU. Fill in session name with something informative. In type, select manual.
For acquisition mode, select accurate hole centering or faster acquisition if aberration free image shift collection is available and desired. Select the desired image format, then select storage folder and EPU will create a directory with the session name. Select the appropriate grid according to which grid hole spacing is being used and press apply.
Select an initial grid square and set an acquisition template. Go to square selection. If all squares are green, click unselect all in the top left.
Open tiles by right-clicking and then selecting open tile. Add or choose a grid square by clicking select, then right-clicking and selecting move stage to grid square. Go to hole selection and press auto-eucentric.
Wait until a grid square image is taken. If the auto-function fails, this may be because the height is significantly off. It can be adjusted manually using the flu screen at grid square magnification.
To measure hole size, move and adjust the yellow circles so they are over the holes with correct size and spacing. Press find holes. Check that the holes have been found correctly.
If not, change the hole size and press find holes again. If this process consistently fails, consider moving to a lower number or brighter spot size at grid square magnification. Use the filter ice quality histogram on the right to adjust hole selection.
This can be useful to exclude areas with thick ice and thin ice. This will be remembered for future grid squares selected during the session. Optimize hole selection with the tools in the select menu at the top.
For example, click remove holes close to grid bar. Go to template definition and press acquire. Click find and center hole.
Change the delay after stage shift and delay after image shift times to one to five seconds, then if available, check maximum image shift value is as desired. Click add acquisition area and click anywhere on the image. Move the acquisition area to the desired location.
Add the defocus range on the top right. A typical defocus list for a membrane protein project is negative 0.8 to negative three micrometer defocus, then add other acquisition areas, arranging them such that areas of acquisition are not doubly exposed with the beam. Click add auto-focus area and click anywhere on the image.
Move the auto-focus area to the carbon surrounding a hole. Standard practice is to auto-focus after centering when using AFIS or every five to 15 micrometers depending on the Z height variation across the square. Click add drift measurement area.
Drift measurement performed once per grid square with a set threshold of 0.05 nanometers per second as a standard setting. The drift measurement area can overlap directly with the auto-focus area. Make sure neither drift nor auto-focus area overlap with an acquisition area.
Go back to square selection and select the squares for acquisition on the grid. Use the number of acquisition areas and expected data acquisition rate to predict how many acquisition areas are required. When all desired squares are selected, press prepare all squares.
Once each square is collected, navigate between the grid squares and fine-tune the holes using the selection brush. Move to a stage location over the specimen and use auto-functions to set eucentric height. Perform microscope alignments using microscope software as described previously and in the text.
Perform coma free alignment within the auto-functions before reinserting and centering the objective aperture and correcting the objective lens astigmatism with EPU. Ensure that both alignments converge on suitable values. Before starting the automated acquisition run, ensure the auto-loader turbo pump is turned off and the objective aperture is inserted if required.
In automated acquisition, press start run to commence automated data acquisition. Using this protocol, broken or dry grids can be screened and discarded at the atlas stage. A gradient of ice is observed across the majority of grids.
During screening, an ideal particle distribution is monodispersed with a range of particle orientations visible. If ice is too thin, it can melt when illuminated with the electron beam, causing excessive motion in micrograph. This is most commonly observed when there is detergent in the buffer.
The ice needs to be vitreous so areas of the grid where the majority of the images show crystalline ice were excluded. Graphical summary of the data was included to assess micrograph quality and to decide if data collection amendments are required. Particle pickers such as crYOLO work sufficiently well for a first pass of the data, enabling progression to 2D class averaging.
Classes which show secondary structure detail should be chosen for 3D analysis while junk particles should be discarded. A subset of particles can be used to generate an initial model for 3D classification and refinement. In the case of RagAB, the dataset contained three distinct conformers which can be separated during 3D classification.
It's important to remember that success at the data acquisition and subsequent image analysis steps are reliant on the obtaining and identifying well-optimized grids during the screening stage. Once a 3D EM density map is obtained, it can be further interpreted through fitting and refining a protein model or building an atomic model de novo. Cryo-EM single-particle analysis enables structured determination of many targets that were not possible previously.
Notably, these workflows have recently been used to solve structures of COVID-19 related macromolecules.
Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.
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