Early junk-particle removal strategy in EMANtomo STA

[Lifei]

Hi,

I have a practical workflow question about early particle cleaning in EMANtomo subtomogram averaging.

If the initial particle stack contains many junk, broken, partial, or mis-centered particles, refinement with a good reference may still pull some of these bad particles into the reconstruction. However, running 3D classification or multi-reference refinement too early can also be unreliable, because particle orientations and shifts may not yet be sufficiently stable. In that case, classification may separate noise, missing-wedge anisotropy, artifacts, or alignment errors rather than true particle quality or biological heterogeneity.

What is the recommended EMANtomo strategy for removing bad particles before 3D classification / multi-reference refinement becomes reliable?

In particular, I would like to ask:

1. Which tools or criteria are recommended for early removal of junk, broken, partial, duplicated, or mis-centered particles?

2. Is it generally better to first perform coarse refinement to stabilize orientations and shifts, and only then run 3D classification / multi-reference refinement?

3. During early classification, should orientations be fixed, locally refined, or re-aligned?

4. How can one diagnose whether the resulting classes are dominated by missing-wedge direction, or other artifacts?

5. For approximately spherical virus particles, how can one avoid mistaking a fuzzy spherical average caused by orientation uncertainty for a valid initial model?

My current understanding is that the workflow should be something like:

initial particle cleaning → initial model → coarse refinement → classification for junk/heterogeneity removal → final refinement

In this view, coarse refinement is mainly a conditioning step: it makes the classification problem better posed by reducing orientation and shift uncertainty. Classification can then be used to clean the particle set and separate possible heterogeneity.

I would appreciate your advice on how to implement this properly in EMANtomo, especially regarding the recommended tools, parameters, and diagnostics for early bad-particle removal.

Best regards,
Lifei

[Muyuan Chen]

I was just writing this today. Maybe it is useful.

https://blake.bcm.edu/emanwiki/doku.php?id=eman2:e2tomo_atpsyn#bad_particle_removal

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