Hi everyone,
I’m working on cryo-EM reconstruction of a membrane protein complex in CryoSPARC, and I’m observing two distinct populations in my 3D refinements. One population has a specific structural feature in the expected orientation, while the other appears rotated ~90° relative to the first.
I’m relatively new to the field and have been trying different approaches to improve resolution. Here’s what I’ve tested so far:
- Template Picking (Curated Exposures)
- Box size 600 → The 90° rotated population appears.
- Box size 700 → The expected orientation is observed.
- Template Picking (Whole Exposures)
- Box size 600 → The 90° rotated population appears.
- Box size 700 → The expected orientation is observed.
- Topaz Training & Extraction (Box 700)
- Ab-initio Classification Results:
- 90° rotated population → 120K particles.
- Expected orientation → 45K particles.
For all refinements, I start with homogeneous refinement (C1 symmetry), followed by non-uniform (NU) refinement with C2 symmetry.
Has anyone encountered a similar issue? Could this be due to flexibility, or is there a processing step I should revisit? Any suggestions on how to resolve or further analyze this issue would be greatly appreciated!