[3dem] RELION 5 Bayesian polishing repeatedly rejects ~50% of particles after successful refinement

[Basil Greber]

We recently had a case where something like this happened because some movies had fewer frames than others (for reasons that we do not understand, but that does not really matter here). Setting the max. frame number in polishing in a way that all movies satisfied that criterion solved the issue. Maybe worth checking.

Basil


> Am 27.04.2026 um 23:50 schrieb Erik Hartwick via 3dem <3dem at ncmir.ucsd.edu>:
> 
> Hello 3DEM community,
> 
> I am reaching out to see whether anyone has encountered a similar issue with Bayesian polishing in RELION 5 via SBGrid. I have run polishing on the same “system” for another project without issue; however, this project seems to be problematic. 
> 
> We are processing an SPA dataset in RELION 5.0.1. Refinement behaves normally, but during Bayesian polishing a very large fraction of particles is consistently removed (~50%). The job completes far enough to report the issue, but polishing repeatedly rejects roughly half of the particles. This was a large collection 17k images at 130kx (​~0.9apix), 300kv, 100A target dimension, preferred orientation is strong, collection was performed with and without tilt, and lastly Blush is being used to help with the orientation bias. 
> 
> What we are seeing:
> 
> upstream particle set refines normally at 1xbin box 256 pix
> 
> Bayesian polishing then removes about 133k particles from a starting set of about 261k
> 
> this has happened repeatedly across multiple attempts
> 
> one representative message was:
> 
> WARNING: 136430 particles in the optics group 1 were removed
> 
> we also saw:
> 
> DEBUG: FC_WEIGHT didn't match
> 
> although this same debug line also appeared in MotionCorr stderr, so I am no longer sure it is directly relevant
> 
> What we have already tried:
> 
> reran MotionCorr from scratch
> 
> reran CTF estimation
> 
> reran micrograph selection
> 
> re-extracted particles
> 
> reran refinement
> 
> traced the lineage backward through the pipeline to look for an earlier metadata issue
> 
> What we found:
> 
> the problem does not appear to be explained by missing micrograph-path matches between the particle STAR and corrected_micrographs.star
> 
> refinement continues to work on these particle sets
> 
> the issue seems to become visible only at polishing
> 
> the problematic lineage appears to emerge after a 3D classification / selection branch, where a parent stack of about ~1M particles was narrowed to a subset of about ~250k particles
> 
> downstream versions of that selected subset continue to show the polishing rejection problem
> 
> A few additional notes:
> 
> this is not normal behavior in our hands, and I have not encountered it before with this workflow
> 
> MotionCorr does report some movies where RELION could not fit a reasonable local motion model and used global trajectory only, but I am not sure whether that is related
> 
> we also saw repeated warnings during extraction about some micrographs having very few or no particles after selection, but regrouping did not appear to explain the polishing rejection
> 
> My main questions are:
> 
> Has anyone seen Bayesian polishing repeatedly remove such a large fraction of particles while refinement still works normally?
> 
> Are there known metadata inconsistencies or STAR-file issues that can survive refinement but cause polishing to reject particles?
> 
> Has anyone seen this associated with a specific 3D classification / selection lineage?
> 
> Are there RELION 5-specific polishing issues with re-extraction, optics groups, or movie metadata that we should inspect more carefully?
> 
> Any suggestions on where to look next would be greatly appreciated. Efforts to start with a new processing attempt have started but knowing why this occurred would be great. 
> 
> All the best,
> 
> Erik
> 
> 
> Erik Hartwick PhD
> Director BioKEM Core Research Facility
> Department of Biochemistry
> University of Colorado Boulder
>  
>  
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