Unexpected difference in FSC from two different microscopes

Helical Processing
1

Hello,

I’ve been using helical reconstruction on a fairly routine filamentous protein from our lab. I’ve previously collected data from a local microscope and using helical refinement with maximal symmetry 8, got this FSC curve:

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The FSC curve reaches 0 like expected.

However using the same grids I collected at a different microscope with a slightly higher pixel size(0.537 instead of 0.54), processed the data in an identical way, and when I attempt to use helix refine with maximal symmetry 8 I get FSC curves like this:

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However when I repeat the process with maximal symmetry not set, the FSC curve returns to normal like so:

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So clearly applying symmetry is causing a duplicate particle affect on the FSC, but why is it suddenly happening now?

Thanks for any insight.

2

For anyone in the future that has the same problem as me, using turning on force redo GS-split parameter seems to have fixed this problem for me. There must have been some issue with the particle distribution in the half-maps.

I don’t know why I had this issue now and not before but at least its an easy fix.

1 Like
3

Sometimes the same effect as duplicate particles is caused by symmetric particles with overlapping fields of view (e.g. because they are crowded).

In either case it could be ameliorated by placing all the particles from a given micrograph into the same half-set.