@chari1 Sounds like you got it. “Relaxation” just means that each particle will always have a chance to be aligned with some weight to each of the symmetry-related poses.
- Refine in Cn first
- Refine/classify in C1 with local search & relaxation(
--sym C1 --relax_sym Cn, same value for “Initial sampling” and “local sampling from”, initial lowpass should be your refine res + 3 A)
You can use a whole particle mask, or a focused mask, but it should include each symmetry related unit. E.g. for SARS-CoV-2 Spike you could use a mask for the whole particle, or one that only included the three RBDs. In this case, I would even make a combo mask that included the “up” or “down” state for each RBD superimposed. But all three subunits should be included, it’s not the same as symmetry expansion.
Breaking the symmetry of the reference (e.g. erasing all but one RBD) may help.
Ref. map absolute scale should always be No (for maps made by Relion itself, you can say yes, but there is no need).
Mask diameter should be the diameter of the particle + 30 A or so (to account for CTF delocalization). This is the same as what cryoSPARC is doing with that 0.85 value at the top of every job type. You can use box size in Angstrom * 0.85 just like cryoSPARC. Smaller values might help if the particle is a lot smaller than the box or it’s very crowded.