[3dem] Abnormal FSC

[Andres Hernandez]

Hi 3DEM,
We have been trying to diagnose the cause of this unusual FSC (sharp loss of correlation at ~4A) and have been unable to find the problem. This results in reported resolutions of ~4.3A when the map quality resembles that of similar ~3.5A reconstructions.

We first thought that it could be due to a limited defocus range, but there is over 1.5 um of coverage in the particles for this reconstruction. I already tried local masks, spherical masks (different positions, radii), 3DFSCs, imposing symmetry, and many other things, but have been unable to identify the cause. The abnormal dip is present with both RELION and Cryosparc workflows.

Any ideas what could be the cause? Has anyone seen an FSC like this before? We think it could be due to the molecule having multiple regions with pseudo-C7 symmetry or D7 symmetry. Imposing symmetry increases the correlation values beyond 4A (~2x) and reduces the dip slightly (~ 0.15 FSC). However, we have gone through great lengths to minimize misalignment, yet localized reconstructions and localized FSCs still show this unusual feature.

[cid:4B99D04C-3145-4014-82CC-50F5A0EE585C at scripps.edu]

Thank you!

Andres
Lander Lab
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