3D ab-Initio reconstruction: alignment issues

I am attempting to reconstruct my highly symmetric protein. While 2D classification yielded nice results, I encountered an issue with unaligned initial ab-initio reconstruction. Any advice on how to address this?
(P.S. I usually enforce symmetry in the refinement process and obtain high-quality particles.)

2D classes:

3D ab-initio:

Yes - try disabling both real space recentering and enforcement of non-negativity. That pair of changes often helps mitigate this phenomenon in highly symmetric cases. If that doesn’t work, you can always enforce symmetry during ab initio.


Disabling did not worked that well, but enforcing symmetry did! Thanks!

1 Like

Try to enforce symmetry during ab initio. And then use C1 for refinement. This workflow also works.

1 Like