Hi, we are observing something which is a bit difficult to interpret, so I wonder if others have seen similar.
Case A. If we don't do any whole movie frame alignment before Relion, then after movie processing and particle polishing inside Relion, the resolution after movie+polishing is better than before it and gets much better after re-refinement of polished particles, all as expected. (All this is after post-processing).
Case B. However, if we do whole movie frame alignment before Relion using Li et al. motioncorr program, then the resolution is good even before any movie processing in Relion but gets worse after movie+polishing. Only after re-refinement of polished particles it gets to be slightly better than in case A. What I don't understand here is why the resolution gets worse after polishing - even if there were no extra movements of particles relative to whole frame movement, b-factor scaling of frames should still help, and b-factors look good, as expected and roughly as they did in case A. We tried to vary SD and Dist parameters in movies processing to limit the extent of particle movement, but that does not seem to help.
I guess it is possible that there are no reliably measurable (by Relion) particle movements apart from whole frame movements in our case and b-factor scaling only has minor contributions. Still, not clear why the resolution gets worse after polishing in case B even when possible particle movements are minimised (although we did not yet try to get them close to 0)?
Thanks for any insights.
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