strategy of refinement for a structure phased by bromide soaking
Dear All, I have a 1.8A resolution data set of a protein collected on a crystal soaked in KBr and I am solving using Phenix. I used AutoSol and gave a rough estimate of f_prime and f_double_prime for the Br scatterer as a starting point. It worked great and Phenix found 31 Bromide ions (only one is fully occupied) and the resulting map is remarkably clear. Automatic building has done a great job and I was able to build most of the 4 monomers present in the AU. However I am confused and have a couple of questions. 1-As Phenix started it generated a fobs_experimental_phases_free_Rflag.mtz file. I have been using this file and no longer my data.hkl file for further refinement. I would like to know why the best_solution after the automatic building run does not contain any of the Bromide ions. It has water molecules but no Bromide ions? What is the reason for that? 2-I have been refining against the mtz file I mentioned but without any of these Bromide ions and although the refinement is converging , I suspect I have been doing something bad and wrong. Should I start my refinement again from the first best pdb after automatic building and include right away the bromide ions with simultaneous f_prime, f_double_prime and occupancy refinement for the anomalous scatterers? As it stands now the Rfree is 22.9 and the Rfac is 19.7. The map looks fine except for the solvent, some of this atoms are obviously bromide and there is extra density in the Fo-Fc around some "solvent" atoms. Thanks a lot in advance for your suggestions. Pascal Egea University of California San Francisco Department of Biochemistry and Biophysics
Hi Pascal,
1-As Phenix started it generated a fobs_experimental_phases_free_Rflag.mtz file. I have been using this file and no longer my data.hkl file for further refinement. I would like to know why the best_solution after the automatic building run does not contain any of the Bromide ions. It has water molecules but no Bromide ions? What is the reason for that?
Since Tom Terwilliger is off-line through Jan 21 I dare to offer a guess: transferring the heavy atoms to the refined model is simply not implemented.
2-I have been refining against the mtz file I mentioned but without any of these Bromide ions and although the refinement is converging , I suspect I have been doing something bad and wrong. Should I start my refinement again from the first best pdb after automatic building and include right away the bromide ions with simultaneous f_prime, f_double_prime and occupancy refinement for the anomalous scatterers?
I think I'd keep the current protein model, but I'd discard all waters and add the Br before continuing with the refinement. I think once the Br are in the PDB file they will survive when you run the wizards. Ralf
Hi,
Since Tom Terwilliger is off-line through Jan 21 I dare to offer a guess: transferring the heavy atoms to the refined model is simply not implemented.
(...)
I think I'd keep the current protein model, but I'd discard all waters and add the Br before continuing with the refinement. I think once the Br are in the PDB file they will survive when you run the wizards.
I'm agree with Ralf. Just to add: once you have added back all Br ions don't forget to run phenix.refine again with ordered_solvent=true option (to restore removed waters), possibly with more than default number of macro-cycles (like 10; default is 3). Also, I would refine individual occupancies for all Br ions and remove those with zero occupancy values after refinement (of huge B-factors). You can do all this in one refinement run. Pavel.
On Thu, 10 Jan 2008, Pascal EGEA wrote:
I would like to know why the best_solution after the automatic building run does not contain any of the Bromide ions. It has water molecules but no Bromide ions? What is the reason for that?
consider input_lig_file_list=my_heavy_atoms.pdb or something like that when running an autobuild. also, there's a couple emails back i asked about a related issue. frankly, i haven't sorted it out. -bryan p.s: always use the latest/greatest phenix.
participants (4)
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Bryan W. Lepore -
Pascal EGEA -
Pavel Afonine -
Ralf W. Grosse-Kunstleve