Unbinned particles giving worse resolution

3D Refinement
1

Hi,

I have a dataset of a membrane protein with 587k particles from 22k micrographs. I did all of the processing with particles binned x2. My binned particles gave a resolution of 3.35A. When I re extracted these particles without binning I now get a resolution of 3.43A.
Does anyone have any idea why it is worse or has anyone seen anything similar? I’m wondering if there are still some bad particles/junk in there that is having a worse effect when unbinned?

My particle is 160kDa with C4 symmetry (4 x 40kDa monomer). I can see from 3DVA that it is quite flexible and it is the flexible regions that are limiting the resolution.

Any thoughts or suggestions would be appreciated.

Thanks,
Niamh

image
P256_J849_fsc_iteration_006_after_fsc_mask_auto_tightening

2

Welcome to the forum @nomalley. What is the pixel size (in Å) before binning?

3

The numerical change of 0.08 Å at the 0.143 cutoff is likely not significant. Are there differences between the maps?

4

Hi, pixel size was 0.65Å before binning.

5

Thanks. I think looking at the maps that the unbinned particles are giving a better map.
I was expecting to see an improvement in the reported resolution but I’m happy if the map looks better. I think the flexibility is causing the main problem.

6

If you haven’t already tried, I would also try running a global CTF refinement (refining beam tilt & trefoil) to see if that might be limiting resolution.

Sometimes when the FSC looks like this (with masked and unmasked FSCs comparable resolution) it can indicate that there is some optical aberration limiting resolution.

I would also try refining beam tilt in image shift groups - if the hardware beam tilt compensation is not well calibrated, this can make a significant difference. We had a recent case where resolution was limited to 3Å until refining beam tilt in image shift groups, which improved resolution to 2.5 Å.

Cheers
Oli

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