[3dem] help with volume alignments

[Toropova, Ekaterina]

Dear 3DEM community,

I've been struggling all day with this problem and admit defeat, would love some help.

I'm trying to do an FSC comparison between my map and a crystal structure, my stumbling block has been to keep the two maps aligned. This is what I've tried.

First approach:
- I've docked the pdb file into my map in Chimera and saved it in the coordinate system of the map.
- I've then used the 'cp from pdb' command in spider to convert the pdb to a spider volume. And here's where I've got a problem - this introduces a rotation, such that when you open the volume in Chimera, it no longer aligns with the EM map.
- I thought I had got around this with a spider RT 3D rotation of 270,180,0 (phi,theta, psi). As it turns out this got me most of the way there - enough to fool me into proceeding with the FSC but on getting lower values than I was expecting I went back to look at the fit and saw that there was still a small rotation and possibly shift that I have spent the last few hours trying to figure out with no avail.

Second approach:
I then decided to try to do this in Chimera using the molmap command to convert the fitted pdb to density, save the map as an mrc file to later convert to spider. This does work - the map stays in the same place as an mrc but when I convert to spider the maps are no longer aligned, there's some discrepancy in the volume center that I can't figure out.

So my questions are:
1) Does anyone know the rotations that need to be applied to a volume generated from the 'cp from pdb' spider command to keep exactly the same orientation as the original pdb when viewed in Chimera?
2) Can I do the FSC comparison with mrc files?

Thanks very much in advance,

Kat



Katerina Toropova
Charles H. Hood Postdoctoral Fellow (Leschziner lab)
Dept. of Molecular and Cellular Biology
Harvard University
305 NWB, 52 Oxford St.
Cambridge, MA 02138



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