[3dem] Abnormal FSC

[Andres Hernandez]

Thanks everyone, It was indeed beam tilt. Beam tilt refinement as part of the CTF refinement in RELION 3.0b0 solved the problem. I gained over 1A in resolution as a result and the FSC looks normal. The documentation only recommends it for structures 3A or better, but it seems to be very useful in cases like this.

On Aug 21, 2018, at 2:21 PM, Wen Jiang <jiang12 at purdue.edu<mailto:jiang12 at purdue.edu>> wrote:

We have encountered a few datasets with similar FSC oscillations and we have identified beam tilt as the source of the problem after refining/testing all particle parameters.

Wen Jiang, Ph.D.
Department of Biological Sciences
Department of Chemistry (Courtesy)
Purdue Cryo-EM Facility<http://cryoem.bio.purdue.edu/>
Purdue University
Hockmeyer Hall of Structural Biology
240 S. Martin Jischke Drive
West Lafayette, IN 47907


On Tue, Aug 21, 2018 at 5:07 PM Andres Hernandez <aherna at scripps.edu<mailto: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.

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

Thank you!

Andres
Lander Lab
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