Re: [ccp4bb] negative density around disulfide bond
On Jun 1, 2014, at 10:08 PM, Eze Chivi ezech...@outlook.com.ar wrote: Hello, when I refine my structure, I see negative density around the disulfide bond. I have 7 copies per ASU, and I can see this density in many of them. In some cases, I see positive density also (negative in the center of the straight line linking S atoms, and positive in both sides). What can I try to solve it? Is it due to radiation damage? Alternative conformation (partial oxidation)? Incorrect disulfide geometry parameters? My resolution is 2.1 A, R/Rfree are around 0.220/0.243, and similar results with refmac5, phenix and PDBREDO. Please find two example pictures in attachment. Thanks for your help! Ezequiel disulfide.jpgdisulfide2.jpg Dear Ezequiel: I apologize if I missed some crucial fact, but the simplest naive explanation would be the disulfide bonds became reduced somewhere along the line, since that would push the non-bonded sulfur atoms apart, relative to where you have them. You should be able to tell with a denaturing gel (run in the absence of DTT or equivalent). I’ve found a bit of Cu(II)-1,10-phenanthroline can help keep the disulfides oxidized. Try re-refining without the disulfide linkages, and see if the difference densities go away. Bill
[ccp4bb] (high) values of R-factors in outermost resolution shell
Dear All, What are reasonable values of Rmerge in the outermost resolution shell? Some of the recent discussions suggest going to those sheels where I/sig(I) ~2 and CC1/2 = 0.5. But I am getting Rmerge Rmeas 1 in the outermost shell for those values of I/sig(I) and CC1/2, and I don't think that makes any sense. Reducing the resolution cut-off while data reduction scaling (aimless) reduces the R-values, but I am not sure how much I should reduce the resolution (if at all). Following are the results from the aimless log file: At a resolution cut-off of 1.62A: Overall InnerShell OuterShell Low resolution limit 42.12 42.12 1.65 High resolution limit 1.62 8.87 1.62 Rmerge (within I+/I-) 0.071 0.019 1.325 Rmerge (all I+ and I-) 0.074 0.020 1.381 Rmeas (within I+/I-) 0.078 0.021 1.446 Rmeas (all I+ I-) 0.077 0.022 1.441 Rpim (within I+/I-) 0.031 0.009 0.575 Rpim (all I+ I-) 0.022 0.007 0.410 Rmerge in top intensity bin 0.031 - - Total number of observations 311022 1839 14876 Total number unique 25311 184 1212 Mean((I)/sd(I)) 19.2 52.7 2.0 Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.740 Completeness 100.0 99.4 100.0 Multiplicity 12.3 10.0 12.3 At 1.6A, the I/sig(I) and CC1/2 in the outermost shell are lower, and the R-merge,meas,pim are higher. looking forward to your suggestions, thanking you, sreetama
Re: [ccp4bb] (high) values of R-factors in outermost resolution shell
That looks good to me Mean(I/sigma) = 2.0, CC1/2 0.74 See endless discussions on this BB about the uselessness of Rmerge as a resolution criterion Phil On 2 Jun 2014, at 09:27, sreetama das somon_...@yahoo.co.in wrote: Dear All, What are reasonable values of Rmerge in the outermost resolution shell? Some of the recent discussions suggest going to those sheels where I/sig(I) ~2 and CC1/2 = 0.5. But I am getting Rmerge Rmeas 1 in the outermost shell for those values of I/sig(I) and CC1/2, and I don't think that makes any sense. Reducing the resolution cut-off while data reduction scaling (aimless) reduces the R-values, but I am not sure how much I should reduce the resolution (if at all). Following are the results from the aimless log file: At a resolution cut-off of 1.62A: Overall InnerShell OuterShell Low resolution limit 42.12 42.12 1.65 High resolution limit 1.62 8.87 1.62 Rmerge (within I+/I-) 0.071 0.019 1.325 Rmerge (all I+ and I-)0.074 0.020 1.381 Rmeas (within I+/I-) 0.078 0.021 1.446 Rmeas (all I+ I-)0.077 0.022 1.441 Rpim (within I+/I-)0.031 0.009 0.575 Rpim (all I+ I-) 0.022 0.007 0.410 Rmerge in top intensity bin0.031- - Total number of observations 311022 1839 14876 Total number unique25311 184 1212 Mean((I)/sd(I)) 19.2 52.7 2.0 Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.740 Completeness 100.0 99.4 100.0 Multiplicity12.3 10.0 12.3 At 1.6A, the I/sig(I) and CC1/2 in the outermost shell are lower, and the R-merge,meas,pim are higher. looking forward to your suggestions, thanking you, sreetama
Re: [ccp4bb] lsqkab problem to be reported to developer
Can you send more details? the log file? the pdb On 30 May 2014 22:54, Carter, Charlie car...@med.unc.edu wrote: This is a bizarre problem. I'm trying to superimpose multiple conformations of the same protein using segments I expect not to change. LSQKAB bails with this error each time: *** RWBROOK error: point code unitfunction ***1 -1022MMDB_F_Atom *** file : 4ARC_rot.pdb *** reason : internal error #2 -- report to developer *** Execution stopped. It also appears very likely that the superposition is wrong. I expect an RMSD for the superimposed atoms (main chain only) of less than 2 Å, yet the program reports that the RMSD is 7 Å. Thanks to anyone for a diagnosis and prescription. Charlie
Re: [ccp4bb] negative density around disulfide bond
Try refining without disulfide bond - from the way density looks it might work. Whether this is what happens in vivo is a different question entirely. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Eze Chivi ezech...@outlook.com.ar /divdivDate:06/02/2014 1:08 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] negative density around disulfide bond /divdiv /divHello, when I refine my structure, I see negative density around the disulfide bond. I have 7 copies per ASU, and I can see this density in many of them. In some cases, I see positive density also (negative in the center of the straight line linking S atoms, and positive in both sides). What can I try to solve it? Is it due to radiation damage? Alternative conformation (partial oxidation)? Incorrect disulfide geometry parameters? My resolution is 2.1 A, R/Rfree are around 0.220/0.243, and similar results with refmac5, phenix and PDBREDO. Please find two example pictures in attachment. Thanks for your help! Ezequiel
Re: [ccp4bb] negative density around disulfide bond
I have seen similar features fairly often when the data collection has been pushed to the limit. The theory is that it is due to radiation damage - you could check by only merging say the first 50% of your data, then seeing if the di-sulphide is intact in those maps. ( wouldnt re-refine much - just set CB-SG occs to 0.00 - recalculate SFs and check the maps) Eleanor On 2 June 2014 07:44, Marjolein Thunnissen marjolein.thunnis...@biochemistry.lu.se wrote: Hi, I would guess radiation damage, see Weik et al., 2000, PNAS 97, 623-628 or for a more recent discussion and thorough overview Sutton et al., 2013, Acta Cryst D69, 2381-2394. best regards Marjolein On 02 Jun 2014, at 07:08, Eze Chivi ezech...@outlook.com.ar wrote: Hello, when I refine my structure, I see negative density around the disulfide bond. I have 7 copies per ASU, and I can see this density in many of them. In some cases, I see positive density also (negative in the center of the straight line linking S atoms, and positive in both sides). What can I try to solve it? Is it due to radiation damage? Alternative conformation (partial oxidation)? Incorrect disulfide geometry parameters? My resolution is 2.1 A, R/Rfree are around 0.220/0.243, and similar results with refmac5, phenix and PDBREDO. Please find two example pictures in attachment. Thanks for your help! Ezequiel disulfide.jpgdisulfide2.jpg *Marjolein Thunnissen* Science Coordinator MX MAX IV Laboratory Lund University P.O. Box 118, SE-221 00 Lund, Sweden Visiting address: Ole Römers väg 1, 223 63 Lund Telephone: +46 766 32 04 17 www.maxlab.lu.se
Re: [ccp4bb] (high) values of R-factors in outermost resolution shell
My suggestion is to ignore Rmerge when making decisions about resolution cutoff. While cc1/2 may still perhaps qualify for the recent discussion tag (is two years recent?), deeply flawed nature of the Rmerge concept is over a decade old. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: sreetama das somon_...@yahoo.co.in /divdivDate:06/02/2014 4:27 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] (high) values of R-factors in outermost resolution shell /divdiv /divDear All, What are reasonable values of Rmerge in the outermost resolution shell? Some of the recent discussions suggest going to those sheels where I/sig(I) ~2 and CC1/2 = 0.5. But I am getting Rmerge Rmeas 1 in the outermost shell for those values of I/sig(I) and CC1/2, and I don't think that makes any sense. Reducing the resolution cut-off while data reduction scaling (aimless) reduces the R-values, but I am not sure how much I should reduce the resolution (if at all). Following are the results from the aimless log file: At a resolution cut-off of 1.62A: Overall InnerShell OuterShell Low resolution limit 42.12 42.12 1.65 High resolution limit 1.62 8.87 1.62 Rmerge (within I+/I-) 0.071 0.019 1.325 Rmerge (all I+ and I-)0.074 0.020 1.381 Rmeas (within I+/I-) 0.078 0.021 1.446 Rmeas (all I+ I-)0.077 0.022 1.441 Rpim (within I+/I-)0.031 0.009 0.575 Rpim (all I+ I-) 0.022 0.007 0.410 Rmerge in top intensity bin0.031- - Total number of observations 311022 1839 14876 Total number unique25311 184 1212 Mean((I)/sd(I)) 19.2 52.7 2.0 Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.740 Completeness 100.0 99.4 100.0 Multiplicity12.3 10.0 12.3 At 1.6A, the I/sig(I) and CC1/2 in the outermost shell are lower, and the R-merge,meas,pim are higher. looking forward to your suggestions, thanking you, sreetama
Re: [ccp4bb] lsqkab problem to be reported to developer
Charlie, Most probably this indicates a problem with your PDB file, either a format or things like misplaced or absent residue name etc. If you can send me your files and exact command that you run, I can have a look into this. In future, if you see a message like report to developer please send it to CCP4 group rather than BB, this will help to deal with it faster. Many thanks, Eugene On 30 May 2014, at 22:54, Carter, Charlie wrote: This is a bizarre problem. I'm trying to superimpose multiple conformations of the same protein using segments I expect not to change. LSQKAB bails with this error each time: *** RWBROOK error: point code unitfunction ***1 -1022MMDB_F_Atom *** file : 4ARC_rot.pdb *** reason : internal error #2 -- report to developer *** Execution stopped. It also appears very likely that the superposition is wrong. I expect an RMSD for the superimposed atoms (main chain only) of less than 2 Å, yet the program reports that the RMSD is 7 Å. Thanks to anyone for a diagnosis and prescription. Charlie -- Scanned by iCritical.
[ccp4bb] phaser issue MR
Dear all, I am using Phaser for Molecular replacement. After Phaser places part of the structure, I would like to run it again, giving this time as search ensembles new pieces of structure. I thought what I had to do was simply to put in know solution the .sol file coming out from the previous Phaser run. However when I do so I get the following message: UNHANDLED ERROR: Program internal error in source file data_pdb.cc (line 144) *** Consistency check (M MOLECULE.size()) failed. *** Please email this log file with some supporting information and/or data to cimr-pha...@lists.cam.ac.uk Could anybody please tell me how to proceed? Maybe there is some option I am not using or some addtional data I need to provide the program... I don't know. Any suggestions are welcome! Thanks a lot in advance! Best wishes, Almu -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
[ccp4bb] Sr. Structural Biology Computing Scientist/Boston
A new position is available in Boston at Harvard Medical School: Senior Structural Biology Computing Scientist Enviroment: The Center for Molecular and Cellular Dynamics Structural Biology Computing Center provides research computing support to 25 structural biology laboratories in the Boston area. We design, implement and maintain a specialized computing infrastructure to support all phases of macromolecular structure determination by X-ray Crystallography, NMR and electron microscopy. The infrastructure includes storage to support data acquisition as well as Linux workstations and OSX research desktops for data processing. We also support specialized research instrumentation computers running the Windows operating system. Job Description: The Structural Biology Computing Scientist position with the Research Computing Core at Harvard Medical School provides a unique opportunity to provide computing support to structural biology laboratories located at Harvard Medical School. Supported groups include laboratories of James Chou (NMR), Stephen Harrison, Stephen Blacklow, Andrew Kruse, Piotr Sliz, Suzanne Walker, Tom Rapoport (X-ray Crystallography), Tom Walz, James Hogle (EM) as well as other groups occasionally utilizing structural biology techniques. You will work as a member of a multidisciplinary research and computing environment that integrates the Computing Core, a software consortium, research laboratories, and teaching initiatives. Your daily responsibilities will include system administration of Linux and OSX workstations and integration of the workstation with research computing services (e.g. storage, authentication, cluster/grid job submission, backups). The individual will be also responsible for support of the specialized structural biology computing workflows. This includes assistance with setting up molecular dynamics computations, assistance with setting up advanced structure determination and analysis pipelines, assistance with preparing macromolecular animations and publication figures and training users. Additional responsibilities will include assistance with software compilation, configuration and customization for cluster deployment including supporting structural biology HPC workflows on clusters and computing grids. Basic qualification: Degree in Computer Science/Bioinformatics; or PhD in Structural Biology; or equivalent combination of education plus relevant research experience. 5-7 years of structural biology research computing support experience. Additional qualifications:Requires an understanding of python or other programming languages; understanding of structural biology applications (e.g. Pymol, COOT, Schrodinger) and structure determination/analysis workflows; strong knowledge of Linux and OSX operating systems. The successful candidate will have excellent organizational skills and particular ability to work independently and prioritize work in an environment of multiple and conflicting interests. Proven project and/or program management skills. Excellent interpersonal and communications skills. Ability to work with discretion. For more information please contactPiotr Sliz.
[ccp4bb] Moire Fringes and Laue Zones
Dear Crystallographers, I have a feeling that Moire finges are the real-space equivalent of the Laue zones in reciprocal-space, and this seems like a very basic idea that must have been explored--anyone know of a source connecting the mathematico-physical dots? Or do the dots not connect? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] Moire Fringes and Laue Zones
Jacob I included a demo using Moire fringes in a crystallography course some years ago. The diagram I used looks just like the second Moire pattern (the annotated one) at this site: http://spie.org/x8449.xml . This is a static Moire pattern; in my demo the two gratings could be translated rotated in the plane relative to each other. Call the horizontal grating the incident beam and the inclined grating the diffracted beam and redefine theta in the diagram as 2 theta (i.e. theta is now the Bragg angle, half the deviation of the diffracted beam). Also call the spacing between successive lines in the gratings (labelled d in the diagram) lambda, and call the spacing between successive Moire fringes (labelled d_m) d. The diagram shows the first order Moire fringe but imagine instead that the spacing d is for the n'th order fringe. Then the equation relating the variables is: n lambda = 2 d sin(theta) It doesn't prove Bragg's law of course, that wasn't my intention: rather it merely demonstrates the geometrical analogy between the Moire pattern and diffraction. The Braggs did actually study Moire patterns and this analogy may well have come to their attention. Is this what you meant? Cheers -- Ian On 2 June 2014 16:05, Keller, Jacob kell...@janelia.hhmi.org wrote: Dear Crystallographers, I have a feeling that Moire finges are the real-space equivalent of the Laue zones in reciprocal-space, and this seems like a very basic idea that must have been explored--anyone know of a source connecting the mathematico-physical dots? Or do the dots not connect? JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] negative density around disulfide bond
Ezequiel, since you mentioned you tried Phenix too: in Phenix you can remove a particular disulfide bond by using a parameter, for example: disulfide_bond_exclusions_selection_string=(chain A and resseq 1 and name SG) or (chain B and resseq 10 and name SG) This works in the command line and GUI. Please let me know (off-list) if you any help with this. Pavel On Sun, Jun 1, 2014 at 10:08 PM, Eze Chivi ezech...@outlook.com.ar wrote: Hello, when I refine my structure, I see negative density around the disulfide bond. I have 7 copies per ASU, and I can see this density in many of them. In some cases, I see positive density also (negative in the center of the straight line linking S atoms, and positive in both sides). What can I try to solve it? Is it due to radiation damage? Alternative conformation (partial oxidation)? Incorrect disulfide geometry parameters? My resolution is 2.1 A, R/Rfree are around 0.220/0.243, and similar results with refmac5, phenix and PDBREDO. Please find two example pictures in attachment. Thanks for your help! Ezequiel
Re: [ccp4bb] negative density around disulfide bond
What does an omit map look like, if you omit both cysteines? Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org Original message Date: Mon, 2 Jun 2014 09:31:28 -0700 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Pavel Afonine pafon...@gmail.com) Subject: Re: [ccp4bb] negative density around disulfide bond To: CCP4BB@JISCMAIL.AC.UK Ezequiel, since you mentioned you tried Phenix too: in Phenix you can remove a particular disulfide bond by using a parameter, for**example: disulfide_bond_exclusions_selection_string=(chain A and resseq 1 and name SG) or (chain B and resseq 10 and name SG) This works in the command line and GUI. Please let me know (off-list) if you any help with this. Pavel On Sun, Jun 1, 2014 at 10:08 PM, Eze Chivi ezech...@outlook.com.ar wrote: Hello, when I refine my structure, I see negative density around the disulfide bond. I have 7 copies per ASU, and I can see this density in many of them. In some cases, I see positive density also (negative in the center of the straight line linking S atoms, and positive in both sides). What can I try to solve it? Is it due to radiation damage? Alternative conformation (partial oxidation)? Incorrect disulfide geometry parameters? My resolution is 2.1 A, R/Rfree are around 0.220/0.243, and similar results with refmac5, phenix and PDBREDO. Please find two example pictures in attachment. Thanks for your help! Ezequiel
[ccp4bb] baverage: no tables were found in this file
Hi CCP4, Recently when I input my pdb file and run the baverage in the ccp4 suit to check the temperature factor, it always tell me No tables were fund in this file. Could you tell me how to fix this problem? Or is there another software instead of baverage I could use to check the temperature factor? Thanks! Bing
