Dear Jacob,
The signal for weak anomalous sites can be stronger in Phaser SAD LLG maps than
in model-phased anomalous difference Fouriers, especially if the substructure
already contains the stronger sites, so that you're just looking for what is
still left to be explained in the SAD data. You could try running Phaser in
MR-SAD mode, giving the current protein model as a partial structure, providing
the substructure of the Se sites, and looking for purely-imaginary scatterers
("LLGCOMPLETE SCATTERER AX" in a script, or check the box labelled "Complete
with purely anomalous scatterer" in the ccp4i GUI). In this situation you
don't want to look for S or Cl atoms as such, because the real part of their
scattering is already accounted for well in the protein model.
Best wishes,
Randy Read
On 1 Sep 2011, at 23:29, Jacob Keller wrote:
> Update:
>
> I tried more anomalous maps, this time with the originally-deposited
> data at 1.8 Ang (mine were similar, substrate-soaked crystals) and
> phases from the refined model, and the Se sites are now ~40-50 sigma,
> and there is still totally nothing at the Cl and S sites, even though
> in 2Fo-Fc the Cl is ~9 sigma, and the S is 8 sigma (the Se is ~15
> sigma). If it has reasonably-high electron density, shouldn't it have
> at least some anomalous scattering? I am wondering whether somehow the
> model phases are biasing the map, but I can't really imagine how that
> would be...
>
> JPK
>
>
> On Thu, Sep 1, 2011 at 3:15 PM, Bosch, Juergen <jubo...@jhsph.edu> wrote:
>> Where in refinement of your model are you ?
>> At an early stage I wouldn't be surprised to only see SeMets but once you've
>> refined your structure and go back to calculate an anomalous map with the
>> improved phases you might double your signal for SeMet and start seeing
>> sulfurs.
>> An alternative explanation, you've blasted your crystals at the synchrotron
>> and the remaining anomalous signal is too weak to show the sulfurs.
>> Just two thoughts,
>> Jürgen
>> On Sep 1, 2011, at 4:03 PM, Jacob Keller wrote:
>>
>> Dear Crystallographers,
>>
>> I recently have been working with a 2.5 Ang SeMet peak wavelength
>> dataset which contains 2 cys's and also a couple of bona fide Cl ions
>> (reasonable b-factor/site is semi-buried/water does not work). In the
>> FFT anomalous difference map using PhiC from the refined model and
>> Dano, I can see the MSE's at ~10 sigma, but no Cl ions, even though Cl
>> should have f" = ~0.3 versus Se's f" = ~4, and no S's in the cys,
>> despite f" = 0.23e. There is really no anomalous peak at all--is it
>> just the smallness of the signal, or are the Se's somehow "swamping
>> out" the other signal? Perhaps the phases are tainted by the presence
>> of semet in the model?
>>
>> Looking for suggestions,
>>
>> Jacob Keller
>>
>> *******************************************
>> Jacob Pearson Keller
>> Northwestern University
>> Medical Scientist Training Program
>> cel: 773.608.9185
>> email: j-kell...@northwestern.edu
>> *******************************************
>>
>> ......................
>> Jürgen Bosch
>> Johns Hopkins University
>> Bloomberg School of Public Health
>> Department of Biochemistry & Molecular Biology
>> Johns Hopkins Malaria Research Institute
>> 615 North Wolfe Street, W8708
>> Baltimore, MD 21205
>> Office: +1-410-614-4742
>> Lab: +1-410-614-4894
>> Fax: +1-410-955-2926
>> http://web.mac.com/bosch_lab/
>>
>>
>>
>>
>>
>
>
>
> --
> *******************************************
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: j-kell...@northwestern.edu
> *******************************************
------
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: + 44 1223 336500
Wellcome Trust/MRC Building Fax: + 44 1223 336827
Hills Road E-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
