[ccp4bb] So many clashes
Dear CCP4 bulletin board I am trying to solve structure with molecular-replacement. I have got good solution using Phaser. The refined structure fits well to electron density and appears reasonable in terms of geometry, ramachandran, rotamers etc. The problem I experience is that there are very many clashes and MolProbity check gives a score of 34th percentile and when I refine, the Rfree does not go below 30% for under 2A resolution. I tried reprocessing in different space group (from P212121 to P21) and also got a MR solution. In P21 number of clashes was reduced but still very high and Rfree was slightly reduced but the gap between Rfree and R was still high. I am not sure what this means and how I can sort out the problem of so many clashes? Any suggestions would be helpful and appreciated regards Careina
Re: [ccp4bb] So many clashes
- look at the clashes one by one and fix them, using your biochemical knowledge and common sense - make sure there are no mistakes in the protein sequence used (resequence if necessary), a few amino acids may be different from what you expect and, combined with local ambiguous density, lead to clashes Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.researcherid.com/rid/B-3678-2009 On 18 Jan 2011, at 10:34, Careina Edgooms wrote: Dear CCP4 bulletin board I am trying to solve structure with molecular-replacement. I have got good solution using Phaser. The refined structure fits well toelectron density and appears reasonable in terms of geometry, ramachandran, rotamers etc. The problem I experience is that there are very many clashes and MolProbity check gives a score of 34th percentile and when I refine, the Rfree does not go below 30% for under 2A resolution. I tried reprocessing in different space group (from P212121 to P21) and also got a MR solution. In P21 number of clashes was reduced but still very high and Rfree was slightly reduced but the gap between Rfree and R was still high. I am not sure what this means and how I can sort out the problem of so many clashes? Any suggestions would be helpful and appreciated regards Careina
Re: [ccp4bb] So many clashes
Dear Careina, Keep in mind that MolPrrobity does not see symmetry (unlike RefDens or WHAT_CHECK). This means that the clashes that may come from having the wrong spacegroup are not detected. Good luck, Robbie Joosten Date: Tue, 18 Jan 2011 10:52:48 +0100 From: mjvanra...@cnb.csic.es Subject: Re: [ccp4bb] So many clashes To: CCP4BB@JISCMAIL.AC.UK - look at the clashes one by one and fix them, using your biochemical knowledge and common sense - make sure there are no mistakes in the protein sequence used (resequence if necessary), a few amino acids may be different from what you expect and, combined with local ambiguous density, lead to clashes Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.researcherid.com/rid/B-3678-2009 On 18 Jan 2011, at 10:34, Careina Edgooms wrote: Dear CCP4 bulletin board I am trying to solve structure with molecular-replacement. I have got good solution using Phaser. The refined structure fits well toelectron density and appears reasonable in terms of geometry, ramachandran, rotamers etc. The problem I experience is that there are very many clashes and MolProbity check gives a score of 34th percentile and when I refine, the Rfree does not go below 30% for under 2A resolution. I tried reprocessing in different space group (from P212121 to P21) and also got a MR solution. In P21 number of clashes was reduced but still very high and Rfree was slightly reduced but the gap between Rfree and R was still high. I am not sure what this means and how I can sort out the problem of so many clashes? Any suggestions would be helpful and appreciated regards Careina
Re: [ccp4bb] So many clashes
If your dataextends to 2A resolution I suggest you run Arp-Warp or Buccaneer to rebuild the structure. At that resolution the automated building programd can usually fix errors. At the end use this option to get the new build back to overlap the original csymmatch -pdbin-ref MR.pdb -pdbin arp-sol.pdb -pdbout arp-sol-overMR.pdb Eleanor On 01/18/2011 09:52 AM, Mark J van Raaij wrote: - look at the clashes one by one and fix them, using your biochemical knowledge and common sense - make sure there are no mistakes in the protein sequence used (resequence if necessary), a few amino acids may be different from what you expect and, combined with local ambiguous density, lead to clashes Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.researcherid.com/rid/B-3678-2009 On 18 Jan 2011, at 10:34, Careina Edgooms wrote: Dear CCP4 bulletin board I am trying to solve structure with molecular-replacement. I have got good solution using Phaser. The refined structure fits well toelectron density and appears reasonable in terms of geometry, ramachandran, rotamers etc. The problem I experience is that there are very many clashes and MolProbity check gives a score of 34th percentile and when I refine, the Rfree does not go below 30% for under 2A resolution. I tried reprocessing in different space group (from P212121 to P21) and also got a MR solution. In P21 number of clashes was reduced but still very high and Rfree was slightly reduced but the gap between Rfree and R was still high. I am not sure what this means and how I can sort out the problem of so many clashes? Any suggestions would be helpful and appreciated regards Careina
[ccp4bb] Postdoctoral researcher in crystallography/biochemistry of transcription complexes
Dear All, We are seeking to recruit a postdoctoral researcher to study multi-protein complexes involved in eukaryotic transcriptional regulation by X-ray crystallography and biochemistry. The successful candidate will join the Christoph Müller group at the Structural and Computational Biology Unit at EMBL Heidelberg. EMBL is a leading international research organization with a collaborative and interdisciplinary atmosphere (see www.embl.de). The Müller group studies molecular mechanisms of eukaryotic transcriptional regulation. Further details are available at http://www.embl.de/research/units/scb/mueller_christoph/index.html. We are seeking to recruit an outstanding candidate with a strong interest in the X-ray structure determination of challenging multi-protein complexes. The candidate is expected to pursue purification, crystallization and X-ray structure determination of transcription complexes in a high-quality team. The successful candidate holds a Ph.D. in structural biology or biochemistry. Previous experience in crystallization/X-ray crystallography is an advantage. The position requires the ability to work independently as well as in a team and excellent technical, organizational and management skills. EMBL is an inclusive, equal opportunity employer offering attractive conditions and benefits appropriate to an international research organisation. Applicants should submit a covering letter describing their motivation, interest in the position and research interests, a CV including a list of publications and the names and contact information of 3 referees. Closing date: February 13, 2011. To apply please go to: http://ig14.i-grasp.com//fe/tpl_embl01.asp?newms=jjid=41162aid=15470 http://ig14.i-grasp.com/fe/tpl_embl01.asp?newms=jjid=41162aid=15470 Please contact me via e-mail if you require additional information (cmuel...@embl.de). Best regards, Christoph - Dr. Christoph W. Muller Joint Head of Structural and Computational Biology Unit EMBL Meyerhofstrasse 1 69117 Heidelberg, Germany email: cmuel...@embl.de phone: 0049-6221-387-8320 fax: 0049-6221-387-519 http://www.embl.de -
Re: [ccp4bb] So many clashes
Hi Careina, Are you using riding hydrogens during refinement? The default in Refmac is to use hydrogens only if present in the input file - change this setting under the Refinement Parameters to generate all hydrogens. This significantly helps with clashes. Also, did you check the quality of your molecular replacement input model? If this model had a lot of clashes, then you are starting with the same problems and it can be difficult to overcome. At better than 2 ang. resolution, one of the automated building programs (arp/warp, resolve, buccaneer, ...) should be able to rebuild much of your model and may help with your clash problem too. good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Tue, 18 Jan 2011, Careina Edgooms wrote: Dear CCP4 bulletin board I am trying to solve structure with molecular-replacement. I have got good solution using Phaser. The refined structure fits well to electron density and appears reasonable in terms of geometry, ramachandran, rotamers etc. The problem I experience is that there are very many clashes and MolProbity check gives a score of 34th percentile and when I refine, the Rfree does not go below 30% for under 2A resolution. I tried reprocessing in different space group (from P212121 to P21) and also got a MR solution. In P21 number of clashes was reduced but still very high and Rfree was slightly reduced but the gap between Rfree and R was still high. I am not sure what this means and how I can sort out the problem of so many clashes? Any suggestions would be helpful and appreciated regards Careina
Re: [ccp4bb] Structures determined: breakdown of methods
On Jan 17, 2011, at 11:46 AM, James Holton wrote: I am willing to bet that the earliest no method entries (particularly the ones that lack a REMark 200 record) were probably MIR, since that was the obvious method to solve a structure for some time. Modern NULL entries seem to be mostly what I call molecular replacement, which includes just refining from a native, etc. not necessarily running an MR search program. I was taught to refer to this method of solution as isomorphous replacement, as long as the space group and/or unit cell had not changed so much that rigid body refinement could not lower the free R-factor enough for conventional refinement to proceed. Diana * * * * * * * * * * * * * * * * * * * * * * * * * * * * Diana R. Tomchick Associate Professor University of Texas Southwestern Medical Center Department of Biochemistry 5323 Harry Hines Blvd. Rm. ND10.214B Dallas, TX 75390-8816, U.S.A. Email: diana.tomch...@utsouthwestern.edu 214-645-6383 (phone) 214-645-6353 (fax) UT Southwestern Medical Center The future of medicine, today.
[ccp4bb] Reminder: Fourth annual CCP4 summer school in USA, at APS, June 7-15
Dear Colleagues, This is a reminder that the on-line applications are being accepted for the 4th annual CCP4 summer school From data collection to structure refinement and beyond, which will take place early June, 2011 at the APS near Chicago. There is no registration fee for the school. The students will be responsible for their own travel and lodging expenses. These and other details (application process, accommodations, site access, contacts etc) can be found at the workshop website at http://www.ccp4.ac.uk/schools/APS-2011/ The school will include data collection, processing, structure solution, model building, refinement, validation, automation of many steps etc. Participants are encouraged to bring their own crystals, raw data or processed data for hands-on problem solving under the guidance of software developers and other experts. Garib, Ronan and Nukri Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov
