Make pseudo-subtomograms

Before processing with any of the regular relion programs (those not specifically intended for tomography, e.g. relion_refine), we first need to construct the individual pseudo-subtomogram particles, equivalent to the particle extraction process in the SPA workflow.

Pseudo-subtomogram particle files are not only related to a particle set. Indeed, they also rely on a tomogram set (tilt series alignment, defocus estimation …) and, in case the motion of particles has been refined, on a trajectory set. Therefore, everytime a Tomo frame alignment or Tomo CTF refinement job-types are run, a new set of updated pseudo-subtomos particles should be reconstructed to keep processing with regular relion programs. This is not necessary in case of specific tomo relion programs (prefix name relion_tomo_) as they are directly fed with original tilt series stack files so referred pseudo-subtomo files are ignored.

In this tutorial, we will first construct the particles in lower resolution, in a binning scale factor 4, to start obtaining a de novo 3D model.

Running the job

Select the IO tab from the Make pseudo-subtomo jobtype. Note that input files can be passed using either an optimisation set file solo and/or the particle set and tomogram set files to override the file within the optimisation set file. Here, we will be using optimisation set files where available.

Input optimisation set:

ImportTomo/job002/optimisation_set.star

Input particle set:

Input tomogram set:

Input trajectory set:

On the Reconstruct tab, make sure the following is set to reconstruct particles with a binning factor of 4:

Box size (pix):

192

Cropped box size (pix):

96

Binning factor:

4

Use cone weight?

No

Cone angle:

On the Running tab, set:

Number of MPI procs:

5

Number of threads:

24

Note that the MPI versions of the tomo relion programs are parallelized in a tomogram base, that is, tomograms will be distributed among the number of processors. Therefore, the Number of MPI processes will not improve the performance if it’s greater than the number of tomograms. Using the settings above, this job took less than 10 minutes on our system.

Your pseudo-subtomo particles and their related CTF and multiplicty patterns will be stored into MRC files in a new directory called PseudoSubtomo/job007/Subtomograms/ separated by tomogram folder. You can check that, together with the updated particles.star file, a optimisation_set.star file is also created.