[Chimera-users] Difference mapping

[Saumya Verma]

Hello everyone!

I am relatively new to cryoEM and am solving my first structure. I have a
question about difference mapping using Chimera, and I’d like to apologize
in advance if it’s too naïve a question for this forum.

I have a cryoEM structure of a mutant virus; the x-ray structure of the
wild-type virus is known. To see the differences between the two
structures, I am following the following steps:
- Deleting the mutated chains from the pdb (so that I’ll be able to detect
these in the cryoEM map) and converting the crystal structure .pdb file to
.mrc, low pass filtering it to the resolution of the cryo-em refined
structure and matching box size and A/pix
- Fitting the two maps in Chimera and subtracting the volumes (using the
command vop subtract)

My question is - how do we decide the resolution to which to lowpass
filter the original structure?

The reason I’m asking is because while my cryoEM structure is 13.3A (FSC
gold standard) (I’ve also checked with ResMap – majority of the voxels are
~14A), it looks very less detailed when compared to the lowpass-filtered
map of the original pdb. And when I’m fitting the two, there are major
differences in the contours, which I think is giving me false differences.

Can someone please let me know what would be the best way to compare the
cryoEM structure of mutant virus with the published structure of the
wild-type virus.

Thank you very much.
Regards,
Saumya
PhD Student
Indian Institute of Technology Delhi, India