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

[Jens Preben Morth]

Dear Pascal

In our experience it was important to slowly extent the phases with  
the keyword res_start 7.0 and s_step 0.02 during density modification  
in resolve, expecially with the combined model experimental phases. In  
case you have a native dataset with no Se, I would consider using it  
too, to get som isomorphous phase information, even if it is only 5-6  
Angstroem, and maybe leaving out the peak dataset. In our case with  
Ta6Br12 the peak dataset was good for finding the sites, but we used  
the high energy remote for refining the sites and final phasing. We  
suspect this could have to do with introduced radiation damage in the  
peak dataset.

good luck
Preben

Quoting "Thomas C. Terwilliger" <terwilliger at lanl.gov>:

> 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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