Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
This could well be due to radiation damage - S are often affected, also Glu and Asp side chains. It is hard to know what to do since the effects are time related. If you have high redundancy maybe you could not use he later batches? Otherwise maybe just relax the B factor restraints and let them show the loss of atoms.. The trouble with that is that you have to relax all side-chain B restraints which may not be so appropriate for ILE say... Eleanor On Apr 4 2012, Chris Meier wrote: MessageDear all, I am refining the X-ray structure of a protein:Data to ~2A were collected at a latest-generation synchrotron.The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms(e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ).Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks,Chris -- Professor Eleanor Dodson YSNL, Dept of Chemistry University of York Heslington YO10 5YW tel: 00 44 1904 328259 Fax: 00 44 1904 328266
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
Hi Chris I would say there's something very wrong if you're seeing -6 sigma difference peaks at O atoms. I don't see how this can be explained by radiation damage. I for one have never seen that before in a structure where there weren't other obvious issues (or maybe I just haven't looked hard enough). I would try refining it with a different program, e.g. Buster, or even a different version of Refmac (I use 5.6.x routinely, but I see there's a 5.7.x now - Garib will no doubt have an opinion on which is the best one to use). At least that will eliminate the software as the origin of the problem: if it doesn't go away then we'll have to think again. Cheers -- Ian On 4 April 2012 16:16, Chris Meier crystallogra...@christophmeier.com wrote: Dear all, I am refining the X-ray structure of a protein: Data to ~2A were collected at a latest-generation synchrotron. The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ). Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks, Chris
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
PS you say the model is complete, but just as important how complete is (are?) the data. -- Ian On 4 April 2012 16:16, Chris Meier crystallogra...@christophmeier.com wrote: Dear all, I am refining the X-ray structure of a protein: Data to ~2A were collected at a latest-generation synchrotron. The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ). Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks, Chris
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
Radiation damage induced loss of definition of disulfide bridges, side chain carboxylates, and certain histidine residues has been observed in synchrotron-irradiated protein crystals. For example, see Weik et al., PNAS 2000, 97, 623. I have also seen a recent paper where radiation damage of a bound protein ligand was apparently observed in a synchrotron beam. I look forward to hearing from others how best to handle this in refinement. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 4/4/2012 11:16 AM, Chris Meier wrote: Dear all, I am refining the X-ray structure of a protein: Data to ~2A were collected at a latest-generation synchrotron. The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ). Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks, Chris
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
I look forward to hearing from others how best to handle this in refinement. Dose-dependent occupancies (tau of an exponential decay function?) refined against unmerged data JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
Hello Chris, Are you refining individual atomic B factors or grouped? Perhaps the B factors of the terminal atoms of the side chain are being restrained to too low of a B factor resulting in excessive negative density? Scott On Apr 4, 2012, at 8:16 AM, Chris Meier wrote: Dear all, I am refining the X-ray structure of a protein: Data to ~2A were collected at a latest-generation synchrotron. The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ). Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks, Chris
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
The PNAS paper you refer to talks about a loss of definition of exposed carboxyl O atoms, i.e. an increase in B factor, but presumably if this is modelled properly then it shouldn't leave a big hole in the difference map. After all, the paper is not claiming that C-O bonds are broken, only that there is increased mobility (or just as likely, induced static disorder). I'm wondering if this is related to too-tight B-factor restraints. I never use the default settings and always use more relaxed ones: in particular I set the weights of B factor restraints across angles to zero, IMO the across-bond restraints are more than sufficient. There has been a historical obsession with getting B factors as low as possible (too-tight restraints will certainly achieve this if that is your goal!), but isn't the true goal of refinement to obtain the model which best explains the data? Cheers -- Ian On 4 April 2012 16:31, Roger Rowlett rrowl...@colgate.edu wrote: PNAS 2000, 97, 623
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
apart from radation damage it could be a combination of: - too tight restraints on the B-factors - 9 sigma not being that much on a the e/A3 scale, i.e. your difference map is very flat (which is good) and the few peaks that remain stand out a lot, even if their absolute height is low... Quoting Chris Meier: Message Dear all, I am refining the X-ray structure of a protein: Data to ~2A were collected at a latest-generation synchrotron. The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ). Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks, Chris Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
Dear Chris Could you please try later version of refmac then if the problem persists please let me know. Before making any suggestions it would be good to make sure that the problem is not related with particular software version (as Ian suggested) regards Garib On 4 Apr 2012, at 16:16, Chris Meier wrote: Dear all, I am refining the X-ray structure of a protein: Data to ~2A were collected at a latest-generation synchrotron. The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ). Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks, Chris Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
could it be that the scattering table would be slightly different for the sulfur atoms at the collected wavelength? Are they Cys or Met residues? if Cys is there a possibility of oxidation to the disulfides?
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
On Wed, Apr 4, 2012 at 10:31 AM, Roger Rowlett rrowl...@colgate.edu wrote: I have also seen a recent paper where radiation damage of a bound protein ligand was apparently observed in a synchrotron beam. That was a manuscript were I would have happily given the coordinates and structure factors to the reviewers with my blessing. Learned a valuable lesson about adopting orphaned data sets though. Cheers, Katherine I look forward to hearing from others how best to handle this in refinement. Cheers, __**_ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 4/4/2012 11:16 AM, Chris Meier wrote: Dear all, I am refining the X-ray structure of a protein: Data to ~2A were collected at a latest-generation synchrotron. The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms (e.g. http://www.dl.ac.uk/list-**archive-public/ccp4bb/2004-07/** msg00532.htmlhttp://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html). Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks, Chris
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
Hi Chris, As has been suggested already, and seems quite plausible to me, it sounds like tell-tale signs of radiation damage. To have little more substance behind this suspicion, some more experimental details could help: What was the dose accumulated during data collection? If the dose cannot be calculated, what was the beam intensity, frame exposure time, number of frames, total rotation of the crystal, crystal size, beam size? If the beam intensity is not known, the beamline and the attenuation factor used might be helpful. What is the space group and how much data were collected? IF you have data with high multiplicity, you may be able to get rid of the latter parts of it maintaining completeness. This would reduce the effects of radiation damage if you are really dealing with it. Alternatively (but again with highly redundant data), you could try zero dose extrapolation. Look up Kay Diederichs' and Dominika Borek's works on this. Regards, 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 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Meier Sent: Wednesday, April 04, 2012 10:16 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] negative difference density around sulphur and oxygen atoms Dear all, I am refining the X-ray structure of a protein: Data to ~2A were collected at a latest-generation synchrotron. The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ). Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks, Chris
