Tomo refinement cycle

We will now describe the specific tomo refinement steps that will refine the particle-specific CTF parameters, tilt angles and beam-induced deformations, which will lead to higher resolution in our subtomogram-averaged structure.

The tomo refinement jobs are CTF refinement and Bayesian polishing, and one tomo refinement cycle would include both. Moreover, for better improvements, we recommend running multiple tomo refinement cycles.

In the current section, we give an overview of how such a tomo refinement cycle would look.

Reference map and FSC data

Before running each of the tomo refinement jobs (CTF refinement and Bayesian polishing), the following are required:

  1. Reference halfmaps obtained by running Reconstruct particle. The implementation of the Reconstruct particle job is consistent with the the implementation of the tomo refinement jobs (e.g. range of the intensities), so it is important to use this reference map rather than maps obtained from 3D auto-refine.

  2. Post-processed FSC data for the reference halfmaps obtained by running Post-processing. This is required to estimate the SNR. If not provided, these programs internally calculate it without phase randomization, so SNR will be slightly optimistic.

  3. Alignment mask file at binning facor 1

  4. optimisation set

Note that, independently of the binning factor of the data in the previous steps, CTF refinement and Bayesian polishing protocols process data in the original pixel size (binning 1). Therefore, the reference map should always be reconstructed at this binning level and proper reference and FSC masks should be used.

Running the jobs

The order of CTF refinement and Bayesian polishing is not important, as long as we keep track of the optimisation set file. In our workspace, in each cycle we ran the two jobs as follows:

  1. CTF refinement, described in detail in the Tomo refinement 1: CTF refinement section, and

  2. Bayesian polishing, described in detail in the Tomo refinement 2: Bayesian polishing section.

3D refinement

After running both tomo specific refinement steps, it is still recommended to run a new 3D auto-refine job to take advantage of the improved tomograms and particles. To this end, we need to construct a new set of pseudo-subtomos and reference maps as described in the Pseudo-subtomograms and reference map at bin 1 subsection. For the new 3D auto-refine job, the same parameters as in the Running the auto-refine job at bin 1 section apply, except for:

On the Reference tab, set:

Initial low-pass filter (A)::

4

On the Auto-sampling tab set:

Initial angular sampling::

0.9 degrees

This new 3D refinement step took just under 2 hours on our system (2 GPU cards) and, after Reconstruct particle and Post-processing with the tight mask, we reached a resolution of 3.6Å, completing the first tomo refinement cycle. After another four full tomo refine cycles, we reached 3.3Å, and depending on the quality of the picked particles, it is also possible to obtain 3.2Å.