[3dem] Abnormal FSC

Scott Stagg sstagg at fsu.edu
Tue Aug 21 14:12:56 PDT 2018

Yes. I think it’s due to cubic ice. That dip is right where the “ring” is in the FFTs of micrographs with cubic ice. I don’t have a fully justified explanation for why it dips, but in my mind it goes something like this: there is a repeating feature at that resolution band, but your refinement isn’t aligning to it, it’s aligning to the particle, so there’s a dip in resolution (apologies for the run-on sentence).


> On Aug 21, 2018, at 5:06 PM, Andres Hernandez <aherna at scripps.edu> wrote:
> 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.
> <FSC-01.png>
> Thank you!
> Andres
> Lander Lab
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