I did run into further difficulties, somewhat related:
I tried to re-extract a subset of particles recentered to align shifts to the unique vertex, but the extracted particles are all centered near the center of the capsid.
The particles input to the Extract From Micrographs job have gone through the following steps:
- HR with sym I
- sym expand I
- VAT - shift ~ 30A along one 5-fold axis
- remove duplicates (4/5 of particles eliminated)
- Subtract signal
- VAT - shift and rotate so vertex is centered with C5 sym
- downsample - crop & recenter using aligned shifts
- 3D classification to select 1/12 of particles with the portal, tail, etc.
- sym expand C5
- 3D classification with sym C1, select class showing 12-fold symmetry of portal and 5-fold symmetry of capsid, with original particle count (ie, fully reversed the most recent sym expansion)
- local refinement C1
- Extract From Micrographs, recenter using aligned shifts
I’ve bolded the steps that I understand to adjust the particle centers and/or shifts. Skipping step 11 and extracting the 3D class that would be input to 11 gives the same result. Extracting after step 8 also gives similar results, particles centered at the positions in step 3.
I’m aware of the issues around recentering during downsampling:
but my expectation is that the shifts applied after downsampling should still be recorded and contribute to the centering of extracted particles. Is that not correct?