[phenixbb] Refining f'/f"?

Leigh Allen c.leigh.allen at gmail.com
Thu May 7 12:39:08 PDT 2009

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?


On Thu, May 7, 2009 at 3:10 PM, Nathaniel Echols <NEchols at lbl.gov> wrote:

> 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.
> -Nat
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