I collected at 1.0809A, which was the optimal wavelength for the beam at NSLS-X29. I will model them as MSEs. I guess the better question to ask is if I should be concerned with not having these negative peaks. Will it negatively affect my R-work/R-free if I continue to work with the data that's not been scaled to keep F+/F- separate?<br>
<br>-Leigh<br><br><div class="gmail_quote">On Thu, May 7, 2009 at 3:10 PM, Nathaniel Echols <span dir="ltr"><<a href="mailto:NEchols@lbl.gov">NEchols@lbl.gov</a>></span> wrote:<br><blockquote class="gmail_quote" style="border-left: 1px solid rgb(204, 204, 204); margin: 0pt 0pt 0pt 0.8ex; padding-left: 1ex;">
<div><div></div><div class="h5">On May 7, 2009, at 2:53 PM, Leigh Allen wrote:<br>
> I'm new to the world of x-ray crystallography. I just solved my<br>
> first SAD structure and I'm on to the refinement stage. The<br>
> difference map generated by phenix.refine has really big positive<br>
> peaks all around my MET residues. If I switch the atoms to MSE,<br>
> then I get large negative peaks. Firstly, I'm not sure if I'm<br>
> supposed to represent these residues as MET or MSE because it's a<br>
> "native," high resolution (1.85) dataset, but it the protein had MSE<br>
> residues. When scaling this data, I did not keep F(+) and F(-)<br>
> separate. The protein's phases were generated using SAD data to<br>
> 2.7A, which using SHARP led to a remarkably interpretable map that<br>
> allowed me to build in the protein by hand. My second question is,<br>
> how should I handle the issue with large + MET/large - MSE peaks?<br>
> Do I need to rescale my data to treat it as anomalous data or is<br>
> there something I can do within Phenix to fix my problem. I tried<br>
> the phenix.refine GUI and set it up to refine f' and f", but it<br>
> appears that nothing really changed.<br>
<br>
</div></div>When you wrote "native", do you mean collected at a wavelength<br>
significantly different than the Se K edge? If it's a longer<br>
wavelength, there will be much less anomalous signal anyway. However,<br>
without separate Friedel pairs I think it is impossible to tell, so if<br>
you want to refine the anomalous coefficients you should rescale with F<br>
+/F- kept separate.<br>
<br>
Regardless of the anomalous signal, if the protein contained Se you<br>
should model the METs as MSEs. I think it's very common for these to<br>
have negative difference map peaks, because they'll undergo radiation<br>
damage very quickly relative to the rest of the protein. If you<br>
collected the high-resolution data set solely to get high resolution<br>
and not phases, this is probably what happened, but I've even seen the<br>
negative peaks around sites used for phasing in a low-exposure dataset.<br>
<br>
-Nat<br>
_______________________________________________<br>
phenixbb mailing list<br>
<a href="mailto:phenixbb@phenix-online.org">phenixbb@phenix-online.org</a><br>
<a href="http://www.phenix-online.org/mailman/listinfo/phenixbb" target="_blank">http://www.phenix-online.org/mailman/listinfo/phenixbb</a><br>
</blockquote></div><br>