FSC model vs map

[Rogelio Hernandez-Lopez]

Hi there,

I am trying to calculate a FSC between my refined model and my experimental map but perhaps I am doing something incorrectly here.

 I tried to use e2pdb2mrc.py to convert my refined pdb into a mrc file with the same pix size than my map. Then, I tried e2proc3d.py to calculate the fsc. It seems that the calculated map has a different coordinate system than my map and I am wondering whether that is affecting my FSC calculations, which look really odd.

How can I create a map from my model in the same coordinate system than my map? Does that affect the FSC calculations?

Thanks in advance

Rogelio

[Steve Ludtke]

Hi Rogelio,

Yes, of course. It is impossible to compare 2 volumes like this unless they are precisely aligned in pixel/voxel-space. ie - if you open both maps in e2display at the same time, they should be precisely aligned. This test does not necessarily work in Chimera, which permits relative shifts between loaded volumes.

To compute the FSC:

1) identical pixel size

2) identical box size in pixels

3) precisely aligned

If alignment is an issue, you can use e2proc3d.py with the --align and --alignref options.

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[Rogelio Hernandez-Lopez]

Hi Steve

Thanks for your reply. It seems that this fixed my problem.

Cheers,

Rogelio

[Md Munan Shaik]

Hi Steve,
I am not sure you are still following this. My question is if want include a mask during FSC calculation using e2proc3d what should be the command. Or if doesnt accept the mask.

thanks,
Munan

[Steve Ludtke]

Is this related to the previous thread, or are you asking a new question?

You can use e2fsc.py, which permits a mask to be specified, but also computes a local resolution map, and optionally locally filtered volumes (ie - it takes a LOT longer to run, but does a lot more, make sure you use threads). 

e2proc3d.py does take the --multfile option which can be used to apply masks, but this can only be used to mask the input volume, not the other volume passed with the --calcfsc option. So, you really need to do this in two stages with e2proc3d. Apply the masks and create temporary files, then compute the FSC between the masked temporary files. 

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Steven Ludtke, Ph.D. <slu...@bcm.edu>                      Baylor College of Medicine 

Charles C. Bell Jr., Professor of Structural Biology
Dept. of Biochemistry and Molecular Biology                      (www.bcm.edu/biochem)

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[Md Munan Shaik]

Hi Steve, 

Thanks a lot for your response. 

I want to generate an FSC plot for map coming from a model vs the EM map. EM map has detergent micelle density so I want to use a mask to exclude micelle during FSC calculation. Map coming from PDB is good and doesn't not mask.

thanks, 

Munan

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[Steve Ludtke]

As long as the model isn't used in generating the mask, this is generally pretty safe. In this situation,

e2proc3d.py map.hdf fsc.txt --multfile mask.hdf --calcfsc frompdb.hdf

should work (untested). If you want to be absolutely sure

e2proc3d.py map.hdf masked.hdf --multfile mask.hdf

e2proc3d.py masked.hdf fsc.txt --calcfsc frompdb.hdf

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Steven Ludtke, Ph.D. <slu...@bcm.edu>                      Baylor College of Medicine 

Charles C. Bell Jr., Professor of Structural Biology
Dept. of Biochemistry and Molecular Biology                      (www.bcm.edu/biochem)

Academic Director, CryoEM Core                                        (cryoem.bcm.edu)
Co-Director CIBR Center                                    (www.bcm.edu/research/cibr)
Co-Director National Center For Macromolecular Imaging                  (ncmi.bcm.edu)

[Md Munan Shaik]

Hi Steve,

Many thanks for your help. 

Thanks,

Munan

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