Sub-tomogram averaging
TO DO. Generating 3D CTF model volumes for sub-tomogram averaging
1. For the sub-tomogram procedure, a tomogram in .mrc format and coordinates of sub-tomograms to be extracted are required along with the input tilt series. You should add to your path all the IMOD commands as well as CTFFIND and the latest version of RELION commands. Coordinates of the centres of the sub-tomograms should be written out in a text file with the syntax - X_coordinate Y_coordinate Z_coordinate
2. First CTFFIND should be run for all images of the tilt series (that was used to generate the tomogram). The results should be written out in a text file with the syntax - Tilt_angle average_defocus_value_from_CTFFIND This can be done automatically using the script run_ctffind.py. Please modify the header values of the script.
3. Now the coordinates from the text file and the average defocus values from CTFFIND will be used to calculate local CTF parameters for each sub-tomogram in each image of the tilt series. This can be done using the script make_ctfstar.py. Please modify the header values in this python script. For each subtomogram, a RELION .star file will be written out. This will be used by relion_reconstruct to generate the 3D CTF volume.
4. Run relion_reconstruct to generate the 3D CTF model volumes. This can be done using the script reconstruct_CTF_volumes.sh that was written out in the last step. This script uses the .star file to read the local CTF parameters and use them to make the 3D CTF model volume.
Particle extraction and sub-tomogram averaging
5. Extract sub-tomograms using RELION as you would do for 2D micrographs. If the image file is a volume and the coordinates values contain X,Y,Z entries, automatically sub-tomograms will be written out rather than 2D particles. If you add --project3d to the RELION extraction command, then 2D projections of each sub-tomogram will be written out.
6. Run "Auto-Refine" as you would for single particles specifying the the CTF volume for each image in a .star file. This can be done using the script prepare_subtomo.py. A sample output is shown in the script.