[phenixbb] How to locate and refine a ligand using anomalous scattering
rjr27 at cam.ac.uk
Wed Feb 9 02:02:51 PST 2011
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".
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.
> 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).
>> 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
>> 1. Locate the bromide position
>> 2. Locate and refine the ligand
>> 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
>> - Using phenix.refine to refine the ligand+protein complex
>> Thank you all for reading this.
>> Structural Biology Department
>> University of Pittsburgh
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Randy J. Read
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