Local refinement mask not aligning

Local Refinement
1

Hi all,

I am trying to locally refine a very large asymmetric complex, with a mask that covers about 1/3 of the protein created using a molmap in ChimeraX, then imported into cryoSPARC v3.3.1 for dilation (5) and soft padding (10). However, the mask won’t seem to align to the correct region of the protein and seems to try to fit the mask into a completely different region.

The input particle stack was from a NU refinement using a volume generated by global 3DVA cluster analysis, and the local refinement alignment parameters are as follows (pose shift/Gaussian priors are switched on with recentering of rotations and shifts each iteration both switched on):

Stdev of prior over rotation: 6°
Stdev of prior over shifts: 2Å
Rotation search extent: 18°
Shift search extent: 6Å

I have double checked the box sizes, and that handedness of the molmap and mask match that of the asymmetric large protein complex I am trying to refine, so any suggestions about what may be causing this would be much appreciated.

Many thanks!

2

Did you resample the molmap (vop resample ongrid) on the grid of the map you are masking?

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3

Thanks your your reply, Oli - yes, I did (volume resample #molmap onGrid #refined map), which normalised the box size to that of the refined map (450³). After doing this, I tresholded the map with vop threshold for minimum and maximum levels before importing.

4

@olibclarke kindly solved the problem for me - I didn’t know it was necessary to resample the molmap onto each new volume to be locally refined. Thanks all!

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