[phenixbb] Refining f'/f"?

Nathaniel Echols NEchols at lbl.gov
Thu May 7 12:10:30 PDT 2009

On May 7, 2009, at 2:53 PM, Leigh Allen wrote:
> I'm new to the world of x-ray crystallography.  I just solved my  
> first SAD structure and I'm on to the refinement stage.  The  
> difference map generated by phenix.refine has really big positive  
> peaks all around my MET residues.  If I switch the atoms to MSE,  
> then I get large negative peaks.  Firstly, I'm not sure if I'm  
> supposed to represent these residues as MET or MSE because it's a  
> "native," high resolution (1.85) dataset, but it the protein had MSE  
> residues.  When scaling this data, I did not keep F(+) and F(-)  
> separate.  The protein's phases were generated using SAD data to  
> 2.7A, which using SHARP led to a remarkably interpretable map that  
> allowed me to build in the protein by hand. My second question is,  
> how should I handle the issue with large + MET/large - MSE peaks?   
> Do I need to rescale my data to treat it as anomalous data or is  
> there something I can do within Phenix to fix my problem.  I tried  
> the phenix.refine GUI and set it up to refine f' and f", but it  
> appears that nothing really changed.

When you wrote "native", do you mean collected at a wavelength  
significantly different than the Se K edge?  If it's a longer  
wavelength, there will be much less anomalous signal anyway.  However,  
without separate Friedel pairs I think it is impossible to tell, so if  
you want to refine the anomalous coefficients you should rescale with F 
+/F- kept separate.

Regardless of the anomalous signal, if the protein contained Se you  
should model the METs as MSEs.  I think it's very common for these to  
have negative difference map peaks, because they'll undergo radiation  
damage very quickly relative to the rest of the protein.  If you  
collected the high-resolution data set solely to get high resolution  
and not phases, this is probably what happened, but I've even seen the  
negative peaks around sites used for phasing in a low-exposure dataset.


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