Two Distinct Populations with Different Density Orientations in Refinement

3D Refinement
1

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:

  1. Template Picking (Curated Exposures)
  • Box size 600 → The 90° rotated population appears.
  • Box size 700 → The expected orientation is observed.
  1. Template Picking (Whole Exposures)
  • Box size 600 → The 90° rotated population appears.
  • Box size 700 → The expected orientation is observed.
  1. 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!

1 Like
2

Hi @F-GST,

Would you be able to post some images of your volumes that illustrate this issue including both the C1 and C2 refinements? Additionally, can you post the cFAR and FSC plots for the refinements as well.

Thanks,
Kye