[phenixbb] How to locate and refine a ligand using anomalous scattering

Randy Read rjr27 at cam.ac.uk
Wed Feb 9 02:02:51 PST 2011


Hi,

You can also use Phaser log-likelihood-gradient completion, starting from your protein model, to find the Br atom(s).  This is usually more sensitive than the traditional model-phased anomalous difference Fourier.

This can be run either from Phaser-EP or from AutoSol.  In Phaser-EP choose the "MR-SAD phasing" mode first.  In either case, you have to choose the data file, a sequence file, and the molecular replacement model.  In AutoSol, make sure that the GUI has identified the PDB file as a "Partial model".

Good luck!

Randy Read

On 8 Feb 2011, at 21:11, Christian Roth wrote:

> Hi Jason,
> 
> I am not an expert, but in my opinion it is enough to collect 1 dataset at the 
> peak wavelength for bromide. Should be anomalous complete for sure. Process it 
> without merging Friedel pairs. Solve the structure via molecular replacement 
> with your known protein structure. 
> Next step ist to create a map using the anomalous differences in your mtz with 
> the phases from your mtz after refinement.
> Now you should see a peak in the density for your Bromine, if ligand is there. 
> Than you have the position of your ligand There should be hopfully density 
> from you normal maps. Put your ligand in it. The bromine dictates the 
> orientation. Put this with the generated library file(phenix.elbow) in phenix 
> and refine it. 
> 
> I think this should work.
> 
> Good Luck and all the best. 
> 
> Christian
> 
> P.S. Despite the fact that you did cocrystallisation, the ligand might not be 
> there, or even at a position which is not the right one. Unfortunately I had 
> already such a case.
> 
> 
> Am Dienstag 08 Februar 2011 19:14:42 schrieb Jason:
>> Hello everyone,
>> 
>> 
>> I have a few crystals to be x-rayed next week. Before that I hope to get a
>> clear idea about what I am doing (I am new to anomalous scattering).
>> 
>> 
>> Facts:
>> 
>> 
>>   1. The crystal is a protein co-crystallized with a ligand
>>   2. The protein structure is known.
>>   3. The ligand has a heavy atom bromide (absorption K-egde=13.47Kev)
>>   4. Data resolution is ~3 angstrom
>> 
>> 
>> Goals:
>> 
>> 
>>   1. Locate the bromide position
>>   2. Locate and refine the ligand
>> 
>> 
>> Questions:
>> 
>> 
>>   1. Do I need to carry out MAD experiment at 3 wavelengths or there is
>>   some other easier way since the protein structure is known (I am not
>>   expecting big change of the protein structure     itself)?
>>   2. Assuming I have the MAD data, what should I do next using phenix to
>>   achieve the two goals listed above? Here are some thoughts:
>>      - Using phenix.hyss to locate the anomalous scatterers
>>      - Using phenix.autobuild to build the protein model (which data set
>> to use?)
>>      - Using coot to add ligands to the protein structure (Is the relative
>>      position between the protein and the ligand known based on
>> phenix.hyss and
>>      phenix.autobuild?)
>>      - Using phenix.refine to refine the ligand+protein complex
>> 
>> 
>> Thank you all for reading this.
>> 
>> 
>> 
>> ======================
>> Jason
>> Structural Biology Department
>> University of Pittsburgh
>> ======================
>> 
> 
> 
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------
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
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Cambridge CB2 0XY, U.K.                       www-structmed.cimr.cam.ac.uk




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