Local motion correction of subparticles?

Motion Correction
1

Hi,

What is the best workflow to perform local motion correction of subparticles (either reference-based or otherwise)?

Ideally, I would like to calculate trajectories from the full particle (which has more signal, and more stable trajectories), but then extract matched sets of subparticles using the improved local alignment parameters and dose weighting.

Currently, the best way I can think of to do this is to align/extract in an oversized box, reconstruct, then use volume alignment tools to recenter, and crop around that center using the downsample tool. However this is (1) very space/time inefficient and (2) fails completely above a certain box size (1126), where reconstruction of the whole particle is currently not possible in cryosparc.

Is there a better way to do this currently?

In a future version, would it be possible to allow a single full-particle reference to be paired with a symmetry expanded particle set after volume alignment tools, to allow for pairing of the sets of subparticles with the appropriate full reference? In either local motion or RBMC?

Cheers
Oli

2

Hi @olibclarke,

Sorry for the delayed response! We haven’t tried this ourselves, but think your workflow should work. As you mentioned, it will likely be space and time inefficient, but to our knowledge would probably be the only way to currently approach this. What box size would you need to use to achieve this for your dataset?

We tried to be careful about units and normalization in our formulation of RBMC, so in principle using hyperparameters from the full particle to motion correct the sub-particles should be okay. Moreover, it seems like hyperparameters that are “in the ballpark” of right tend to give reasonable results. That said, less signal is fundamentally harder to align, so I don’t know if that effect would manifest to a significant degree. If you do attempt to do this, please let us know how it goes.

Additionally, we have noted a feature request for sub-particles and symmetry expanded mates in RBMC.

All the best,
Kye

1 Like
3

Hi Kye,

The problem is that the native box size I would need for this would be on the order of 1400 pixels - and I would then need to refine before cropping the subparticles out, as otherwise the shifts will be inaccurate (due to drift during RBMC, as discussed elsewhere).

I guess I could run RBMC, downsample 2x & refine to get “good enough” alignments, then crop out subparticles from the original RBMC particles using the alignments from the downsampled refinement? as otherwise refinement will fail in the original box size…

But there is another problem, I think - in order to start RBMC, I need to provide a matching set of particles & volume - but I can’t reconstruct from 1400px particles as I will run out of GPU RAM. I guess I can just extract the particles in that size, and crop/pad the volume from a smaller box size to match? EDIT: actually just tried this, and it requires one additional step - because Vol Tools only works on one map (not all the passthrough maps), separate vol tools jobs need to be run for the full map and both half maps, and then the outputs recombined using the lower level slots. EDIT2:… but when I try this, it doesn’t work :frowning: see screenshot

Cheers
Oli

image
image1802Ă—340 54.3 KB

EDIT3: I tried a different approach - performing the pre-RBMC reconstruction directly in a reduced box size - but this fails with EWS correction on:

Traceback (most recent call last):
  File "cryosparc_master/cryosparc_compute/run.py", line 115, in cryosparc_master.cryosparc_compute.run.main
  File "cryosparc_master/cryosparc_compute/jobs/refine/newrun.py", line 1174, in cryosparc_master.cryosparc_compute.jobs.refine.newrun.run_homo_reconstruct
  File "cryosparc_master/cryosparc_compute/engine/newengine.py", line 3279, in cryosparc_master.cryosparc_compute.engine.newengine.reconstruct
  File "cryosparc_master/cryosparc_compute/engine/newengine.py", line 3292, in cryosparc_master.cryosparc_compute.engine.newengine.reconstruct
  File "cryosparc_master/cryosparc_compute/engine/newengine.py", line 482, in cryosparc_master.cryosparc_compute.engine.newengine.EngineThread.setup_ews_correct
AssertionError: TODO must check box size convention for ewald sphere radius calculation