[phenixbb] effect of solvent fraction on density modification/solvent flattening for a membrane protein

[Thomas C. Terwilliger]

Hi Pascal,

It sounds to me as though there is indeed 1 molecule and 6 sites to be
found in your structure.  The autosol wizard may look for 14 sites but if
there are only 6 good ones it should stick with those, as it seems to have
done.

The solvent fraction does affect the density modification step. However
the difference between using a value of 50% or 70% will make only a small
difference in the final phases, as the solvent boundary mask is a
probabilistic one, so that there is no sharp cutoff at 50% or 70% of the
asymmetric unit.

If your final map shows helical density, then you might try running the
AutoBuild wizard with the flag helices_strands_only=True . This forces the
building to stick with secondary structure, which I am guessing is all
that it will be able to build of your model. The model-building of the
secondary structure with this flag set can be much better than standard
secondary-structure model-building with the AutoBuild wizard at lower
resolutions such as yours.

All the best,
Tom T

> Dear All,
>
> This is a question about the effect of solvent fraction estimation on
> the quality of the initial phases.
> I am trying to solve the structure of a membrane protein at moderate
> resolution 3.5A from a MAD data set (peak, inflection and high
> energies).
> I have one copy of my complex which gives me a solvent fraction of
> 0.7 without considering the contribution of the detergent.
> With a related molecule I found a Phaser-MR solution but it does not
> refine although it is right.
> I am using Phenix with the phases obtained from molecular replacement
> and combining them with the MAD signal to locate the seleniums. A
> visual inspection of the anomalous fourier difference map shows 6
> sites out of the 7 expected at 4sigma  contouring.
>
> If I let Phenix decide by itself it goes on its own with  two copies
> in the ASU and considers a solvent fraction of 0.50; then it finds 7
> sites (and not 14 ? why is that?) (the seventh is weak but we expect
> this for this region of the complex).
> now if I force Phenix to take ncs_copies=1 and solvent fraction=0.7
> then it only find 6 sites.
>
> Wether it is 2 "freely chosen" molecules/ASU and 0.5 of solvent  or 1
> "forced" molecule/ASU and 0.7 of solvent, the 6 first sites are
> identical (although occupancies are quite higher in the first case)
> Should I take into account the detergent and lower somewhat my
> solvent fraction (in the 0.6 range maybe) with the fixed one copy ASU
> estimation or just trust Solve/Resolve when it runs the density
> modification part. Does it really affect the quality of the phasing?
> Between these two extremes I am surprised to see that the quality of
> the resolve map are quite similar apparently showing clear new
> structural features.
>
> Thanks in advance for your comments,
>
>
> Pascal F. Egea, PhD
> Post Doctoral Researcher
> University of California San Francisco
> Department of Biophysics and Biochemistry
> Robert Stroud Laboratory
> pascal at msg.ucsf.edu
>
>
>
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