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
