fsc plot

[maria]

Dear EMAN2 developers, I'm a computational structural biologist. I want to compare the experimental electron density map of a protein system with an electron density map generated from an ensemble of pdb structures of my system. in order to do that I did the following steps: EMAN 2.99.47 ( GITHUB: 2023-03-04 13:49 - commit: 3f313008c3185410fe859663e763dffb9c0b6fcc ) Your EMAN2 is running on: Mac OS 10.15.7 x86_64 Your Python version is: 3.9.16 1) e2iminfo.py -H experimental_map.mrc 2) e2proc3d.py experimental_map.mrc experimental_map.hdf 3) e2pdb2mrc.py ensemble.pdb ensemble.mrc --apix 0.73 --res 3 --box 256 --center 4) e2proc3d.py ensemble.mrc ensemble_ali.hdf --alignref experimental_map.mrc --align rotate_translate_3d_tree 5) e2proc3d.py experimental_map.hdf fsc.txt --calcfsc ensemble_ali.hdf This generate the fsc plot that you can find attached to this email (FSC.png). If I compare it with the one present in a publication of the same system (see the FSC_PAPER.png plot attached), the trend is quite the same, but in my plot I have some steep picks and I'm not understanding why. I also tried to generate masks, tight and loose, from a single pdb of the ensemble and sum it to both the maps and then calculate the fsc plot, but the results were pretty the same (SEE FSC_MASK.PNG). 

The result is also similar if I generate the map thought a single pdb structure instead from the ensemble (see FSC_PDB_ENS.png). I'm doing something wrong? which step I'm missing?

Best regards,

Maria

[Muyuan Chen]

I would guess it is some small misalignment…

1. Make sure the model fit into the map to begin with, and there isn’t too much unmodeled density. 

2. Make sure the apix you specify is the actual pixel size of the map. 

3. In theory if the model fits in the map, you don’t need the “—center” for e2pdb2mrc, and don’t need the alignment step either. However the mrc origin might make things complicated. I would use “—align translational” instead of rotate_translate_3d_tree to avoid the extra interpolation. 

Muyuan

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<FSC_PDB_ENS.png><FSC_MASK.png><FSC.png><FSC_PAPER.png>