April 2012 - phenixbb - phenix-online.org

links in phenix summary
by Christian Roth 04 Apr '13

04 Apr '13

Dear all, thanks to the phenix team which helped me a lot to resolve all issues regarding the correct link definitions. I have summarised a few things which one might consider if nonstandard links are necessary. Try phenix ready set to get your links out of the pdb. Do not forget to change the possible LINKR to LINK! Inspect the links and decide if they are useful! Peptide links are automatically incorporated if residues are close (inspect geo file) Non standard peptide links could be created using using an now available alpha release script: elbow.ligand_linking model.pdb Inspect the generated cif file and correct angles and incorporate dihedrals and so on if necessary. Sugar links which exists in the mon_lib.cif should be incorporated using the apply_cif link option like refinement.pdb_interpretation.apply_cif_link { data_link = ALPHA1-4 residue_selection_1 = chain B and resname GLC and resseq 1 residue_selection_2 = chain B and resname GLC and resseq 2 } According to the link definition the C1 oxygen of the following sugar with the higher number must be removed. The automatic incorporation of sugar links will be probably soon available in phenix. Nigel is already working on this. If one want use hydrogens in the refinement, they have to be added previously with ready_set and afterwards the OH4 sugar hydrogen has to be removed before the refinement. For links with symmetry related atoms the symmetry operator must be included in a custom bond definition efinement.geometry_restraints.edits { bond { action = *add delete change atom_selection_1 = chain A and resname HEM and resid 154 and name FE atom_selection_2 = chain A and resname MTO and resid 155 and name O symmetry_operation = -x,y,-z distance_ideal = 2.2 sigma = 0.01 } } The operator can be obtained using iotbx.show_distances your.pdb > all_distances Search for keyword "sym" or from Coot clicking on the symm option. Coot print out the operator and the translation term separately. Both has to be merged in the final operator used in phenix! Christian

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Re: [phenixbb] Geometry Restraints - Anisotropic truncation
by Leonid Sazanov 03 May '12

03 May '12

Hi, so will we see now the possibility to apply anisotropic resolution limits truncation in Phenix? Despite the fact that a lot of people use UCLA server, there is still no possibility to do this in any major crystallographic software, AFAIK. Phaser does the scaling, but no truncation, and noise sitting in those regions where there is no signal, is a real culprit. Large SIGFs there do not resolve the problem completely. Thanks. >>>>>> Message: 4 Date: Wed, 25 Apr 2012 09:05:57 -0400 From: Mario Sanches <mariosan(a)gmail.com> To: PHENIX user mailing list <phenixbb(a)phenix-online.org> Subject: [phenixbb] Geometry Restraints - UPDATE .......... It turns out that the reason to my nightmares was a highly anisotropic dataset. When I tried to fix geometry related problems in coot, phenix.refine was twisting the geometry back again, probably because it was trying to fit a lot of noise due to the anisotropy. Everything was getting worst, geometry, clashscore, and Ramachandran. Pavel finally corrected it by using this server: http://services.mbi.ucla.edu/anisoscale/ ............ -- Dr. Leonid A. Sazanov Research group leader Medical Research Council Mitochondrial Biology Unit Wellcome Trust / MRC Building Hills Road Cambridge CB2 0XY WEB: www.mrc-mbu.cam.ac.uk Tel: +44-1223-252910 Fax: +44-1223-252915

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How to include restraints when refining DNA?
by Xun Lu 03 May '12

03 May '12

Dear all, I am refining a protein-DNA complex structure at 2.9A resolution. The R/Rfree is 0.2181/0.2501, which is pretty good, but I can tell that the DNA is not well refined. Some sugar puckers are not right. The distances between some base-paired bases are kind of short (~2.5A). I've tried including a reference model for the DNA, or including a cif file, and I've also tried to turn on the secondary_structure_restraints.... nothing worked. I must have not done it in a correct way. But how to do it? And how to deal with sugar puckers? In refmac, I can set the distances between bases during the refinement, so there must be a way to do it in phenix as well? Any advice is welcome. Thanks! Xun -- Department of Molecular and Structural Biochemistry North Carolina State University

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Re: [phenixbb] Scaling of diffraction data
by jens j birktoft 02 May '12

02 May '12

Hi Everybody, Is there a protocol for re-scaling several sets of diffraction data against a common reference set, either native or based on a Fcalc set computed from a common input model. The are from a number of derivatives, are all isomorphus, and are anomalous data . We want to compare the relative peak heights in Fo-Fc and Anom. difference maps Cheers Jens +++++++++++++++++++++++++++++++++++++++++++++++++++ Jens J. Birktoft e-mail: birktoft @ nyu.edu <jens.knold(a)gmail.com> ; Phone: 212-749-5057 +++++++++++++++++++++++++++++++++++++++++++++++++++

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Inputting DNA for Phenix MR and/or Autobuild
by Charles Allerston 02 May '12

02 May '12

Dear all, I can't find much in the way of documentation for this. Can someone point me to some please? I basically want to know the correct format for entering DNA into the search for MR and inputting into Autobuild. If I have a double stranded DNA do I enter it as >DNA1 AATT:AATT >DNA1 AATT TTAA etc. ? cheers, charlie Dr. Charles Allerston Genome Integrity & Repair Structural Genomics Consortium Nuffield Department of Medicine Old Road Campus University of Oxford OX3 7DQ http://www.thesgc.org/scientists/groups/oxford/genome-integrity-and-repair

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Estimation of ligand binding affinity by occupancy refinement
by Zoran Stefanic 01 May '12

01 May '12

Dear all, I have a question about sensitivity of occupancy refinement in phenix. I have a series of datasets done at our home instrument of relatively modest resolutions from 2.8 to 3 A. These are performed on crystals soaked in progressively higher ligand concentrations. We are trying to see to which of the several active sites the ligand binds first so we went from low to high ligand concentrations. We wanted to estimate the amount of ligand bound by refining the occupancies of the ligand. We expect one active site to bind more strongly than the other two active sites. This was indeed what we observed but when we refine the occupancies of the ligands in weaker binding sites starting at 0.2 occupancies they rise to 0.5-0.6 although some atoms are totally out of density and some are in. B factors are reasonable, i.e. don't go wild. My question would be: how reliable is this estimation of ligand binding by occupancy refinement? Why doesn't the occupancy of ligands stay low if afterwards the density fit of the ligand looks rather unconvincing? Thank you in advance Zoran -- Dr. Zoran Stefanic Division of Physical Chemistry Rudjer Boskovic Institute Bijenicka 54 P.O. Box 180 Zagreb Croatia Phone: 00 385 1 457 1203 Fax: 00 385 1 468 0245

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metal coordination
by Panne Daniel 30 Apr '12

30 Apr '12

Hi all, I had a question concerning metal coordination (zinc in this case). I have used phenix.metal_coordination to generate geometry restraints for refinement. However phenix.refine does not write out the necessary LINK records into the PDB file. This seems to lead to problems later during validation, as the metal coordination is not properly recognized. What would be the best way to generate the LINK records in Phenix? Thanks, Daniel

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Error of Sculptor
by Zhang yu 26 Apr '12

26 Apr '12

Dear Phenixer, When I used Sculptor to generate a new model, there is a error which I can not figure out. I input a PDB file (*.pdb) and an alignment file (*.aln). The following is the log file (contents in ######). There seems something wrong with the *.aln file. ################# Reading PDB file ---------------- File name: **/**/******/******/**.pdb Separating solvent from macromolecules... Reading alignment files ----------------------- 1. **/**/**.aln ##################### The following is the content of the alignment file: **_** MVYSYTEKKRIRKDFGKRPQVLDVPYLLSIQLDSFQKFIEQDPEGQY----GLEAAFRSV **_** ---------MEIKRFGRIREVIPLPPLTEIQVESYRRALQADVPPEKRENVGIQAAFRET * **: :*: :* * .**::*::: :: * : *::****.. What is the problem with the alignment file? Thank you in advance! Fish

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cut-out mtz edition
by Dialing Pretty 26 Apr '12

26 Apr '12

Dear All, I just cut out a fragment of chain and the corresponding electron density from the whole molecule. Will you please give me some suggestion on how to edit the attached file so that those electron density not from the protein fragment can be erased? Or do you have other methods to produce high quality figure which just shows the fragment chain and the fragment eletron density? Cheers, Dialing

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Geometry Restraints - UPDATE
by Mario Sanches 25 Apr '12

25 Apr '12

Last week I asked a question about geometry restraints at low resolution. I would like to thank you all that took the time to answer, specially Pavel for looking at the data. First, here is the original question: __________________________________________ I am refining a low resolution structure (2.9A) using phenix.refine. As I continue the refinement Rwork/Rfree are dropping but my geometry statistics in general are very bad. My clashscore is getting worst and the RMS(angle) is really high. I have tried to play with the "wxc_scale" parameter and also ran phenix selecting the "Optimize X-ray/ADP weight" option. All attempts end with a RMS(angle) of ~3.8 and a Clashscore of ~190. Is there a known procedure to deal with this? I feel that my geometry weighing term is too loose but I don't know exactly how to tighten it other than decreasing the wxc_scale parameter. I have gone and manually corrected bad positions before but given the low resolution nature of the structure the clashes tend to be reestablished during refinement. Thank you in advance for your help, __________________________________________ It turns out that the reason to my nightmares was a highly anisotropic dataset. When I tried to fix geometry related problems in coot, phenix.refine was twisting the geometry back again, probably because it was trying to fit a lot of noise due to the anisotropy. Everything was getting worst, geometry, clashscore, and Ramachandran. Pavel finally corrected it by using this server: http://services.mbi.ucla.edu/anisoscale/ I should have been more diligent regarding that, since I could clearly see that my data was anisotropic from the diffraction images. I just didn't know how important this parameter could be. Anyways, phenix.xtriage will tell you if your data is anisotropic. The lesson is to pay attention to it. In my case, the quality of the electron density improved considerably, the geometry improved massively (manual intervention in coot was absolutely necessary, but now it stays correct after I manually fix the problems), clashscore lowered, etc. As a side note, it worked better without hydrogens in my case than with. It is certainly a parameter to try if you are having problems. Thank you all again for the tips. Mario Sanches -- Mario Sanches Postdoctoral Researcher Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave Toronto - Ontario Canada M5G 1X5 http://ca.linkedin.com/in/mariosanches

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