Then did abinitio(3 class and 2 class). Ab initio didnt generate good model, so I imported a apoferritin model from EMDB and used it as one of the input volume in heterogenous refinement. This time I got a pretty good looking structure at 4.06A.
But when I did NU refinement of this structure, resolution decreased to 6.22A. I have used default settings in all the job type. I have total 75,570 particles.
Your 2D classes looks great and I don’t think you need to spend more time removing junk right now. I would focus on making ab-inito work, with the particles you have, instead of importing 3D volumes as references.
The easy way to make ab-initio work is probably to enforce the O symmetry of Apoferritin. 2 classes from 10k particles is usually plenty.
If you want to make ab-initio work in C1, while the particle has high symmetry, you could try changing a few settings:
Change the “Maximum Resolution” from 12 Å to ~8 Å.
Turn off “Center structures in real space”.
Turn off “Enforce non-negativity”.
Either way, once you have generated a good ab-initio volume to refine agains, you should use that O symmetry during your homogeneous (or NU) refinement.
