This protocol provides an easy access route into the increasingly popular but complex world of cryo electron tomography. Six cellular specimens can be imaged. Institute in a close to native state.
Micromolecules can then be resolved at high resolution in their native cellular environment by using CROW, ET and sub atom averaging. Begin by starting the Tomo five software from the TEM server pc. Start the session set up by adjusting the image acquisition parameters in the preparation tab, under presets.
Copy the parameters of image acquisition to all the other high magnification presets. Press Set"to set exposure to the microscope and Get"to get the exposure settings to all other high magnifications after selecting them individually. To collect an atlas, click on the New Session"in the Atlas"tab.
Set the session preferences. Enter the storage path and the output format and press apply. Select Screening"and tick all the atlases to be acquired.
Select Close Call Valves"If the microscope is unsupervised this will close the column valves. Then to start the screening, press the start button. Inspect the single or multiple atlases for targets by clicking on the grid in the left panel under Session Setup"Then left click and drag the mouse to move around and middle scroll to zoom in and out.
Choose the grid for the target setup. Select it and click on Load Sample"from inside the software. To perform image shift calibration, in the Auto Functions"tab set the preset to Eucentric Height"Navigate to Auto-Eucentric"by stage tilt, and press start.
If the feature remained centered during targeting skip the image shift calibrations. Otherwise, go to the Preparation"tab for calibrating image shift select Calibrate Image Shift"and press start. This will align lower magnifications to the feature centered at exposure magnification.
For tomography set up, start a new session in Session Setup"for biological samples. Choose Slab like"as the sample type and select Batch"and Low Dose"Then select the Output format and storage"folder. Optionally, add an email recipient and press Apply"To set up targets, Go to the atlas arrow, find a region of interest and move by selecting the options that pop up with a right mouse click.
Take an overview image to confirm a good position for Eucentric height adjustment. Then press autoeucentric"to run the eucentric by stage tilt routine, reacquire a new overview image to update the eucentric height. Inspect the square overview or the acquired search map.
Move to a region of interest and press acquire search. Then inspect the search image if the region of interest is not centered, right click at the desired position and repeat Move stage here"and acquire image. Adjust the focus and tracking areas.
Left click to drag the tracking and focus areas. Once all the parameters are set press Add Position"To perform auto functions check the settings to perform the alignments via the Auto Functions"tab by navigating the atlas to an area of carbon. Bring that area to eucentric height and follow the order of alignments as described in the text manuscript.
Next, start the automated acquisition of the tomography tab. Select the Data Acquisition"slab out and set the desired parameters. Set up data acquisition parameters:tilt step, maximum positive angle, maximum negative angle, tracking scheme.
Then select Close Column Valves"for the eucentric height scheme. Start with preparing input files and directories. Establish a project folder under the Project"folder.
Make another new folder fixed stacks and prepare the input files. Tilt series one fixed tilt series 1. XF and tilt series 1.tlt.
To calculate the defocus, update the parameter file with the microscope and imaging parameters. Copy a parameter file to the project folder. Change its name to param_CTF.
M and run the indicated command. Next, check the CTF estimation results. For each stack, check the aligned stacks using 3D mod and ensure that the fiducial beads are erased correctly.
Ensure that the handedness is correct by running the indicated command. Then check the defocus value and ensure it matches the theoretical CTF estimate. To define the subregions, generate a bend tomogram.
In the project folder, run the indicated command. Determine the boundaries by choosing six points Xmin Xmax Ymin, Ymax, Zmin, and Zmax, to create one subregion under the folder bin10, run the indicated command. Next, run particle picking for each subregion.
For the apoferritin dataset carry out a template search at bin six. Modify the template underscore angle. Search parameters to determine the angle, range and intervals for in plane or out of plane search in degrees.
In the project folder run the indicated command. Remove the incorrect particles using 3D mod under the folder.Convmap_wedge_Type2_bin6. Run the indicated command.
Next, initialize the project. In the project folder run the indicated command to create a database for EM clarity, AppoF.mat. Perform tomogram reconstruction before sub tomo averaging and alignment to generate CTF corrected subregion tomograms at bin4, run the indicated command.
Next, to perform sub tomo averaging and alignment, carry out the averaging using the CTF corrected sub telegrams starting at bin4. In the project folder, run the indicated command. Continue to do alignments and run the command.
Clean the overlapped particles by running indicated command. Perform final reconstruction by combining the two half data sets. In the project folder run the indicated commands.
Overview of the tomography workflow for the cellular and molecular tomography are shown here for cellular and lamella samples, the data collection strategy largely depends on the sample and the goal of the imaging study. Collection parameters for several cryo ET studies are shown here. Representative tomograms of molecular samples such as apoferritin, thin cellular processes, and focused ion beam milled lamella of the thick cellular specimen are shown here.
High resolution structural analysis can help to reveal binding sites for drug targets and tomography and sub-tomogral averaging have also been aiding vaccine development against SARS-CoV-2.
