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I think you have been caught by a new REFMAC "feature" which tries to design its own TLS groups including linked H2Os and ligands. Check your tls output records and see what it has clustered into a group.. I am not sure how to disable this - at times I want to override any automatic selection.. Eleanor On 03/01/2011 07:48 PM, Judith Reeks wrote: > Dear All, > > Thank you for your suggestions. Many of you asked what the occupancies > were in the region and they were all one, so partial occupancy was not > the problem. > > I was using TLS restraints during the refinement when this problem > happened. Given the suggestions that TLS may be a problem and that might > be causing the low B-factors, I went back and re-ran the refinement > without TLS and the problem disappeared. Then I submitted my latest file > to the TLSMD server for new restraints and the next round of refinement > got rid of the problem. The B-factors increased to normal levels (~15 > compared to ~5 before) so it seems to have done the trick. > > Thank you to everybody for their help, > > Judith Reeks > > [log in to unmask] > > School of Chemistry > > University of St Andrews > > > On 01/03/2011 17:28, Mark Robien wrote: >> Hmm - kinda interesting >> >> In addition to the sorts of things suggested by Mark van Raaij, older >> versions of Refmac >> were prone to have a phenomenon with B factors - once they get over a >> certain level, the algorithm >> has a very hard time bring them back down, even when the data suggests >> it. >> >> I've usually seen it with much higher B factors than you seem to have >> here - for example, loops where the B's >> are actually 40-60, but previous rounds of refinement have the B >> factors 60-80 or higher. >> Judging from the coot screen & the residues you are focusing on, I >> doubt that is the answer (unless you also >> have a TLS model, in which case I'd wonder). If you do have TLS - >> well, things get more complicated; for example, >> is this the edge of a TLS domain? >> >> Nonetheless, you could try the solution for the problem that I >> describe - which is to reset all the B factors to a (very) >> low B factor - maybe even as low as 2.0 (lower?), and then another >> round of refmac - with a sufficient number of >> cycles - will re-refine the B (and xyz), thus escaping the region of >> refinement space that has a very weak gradient. >> >> A variant approach that might be appropriate - similarly reset the B >> for the (?small) region of your model that has the problem. >> >> Sadly, it's been awhile since I did any refinement myself - but the >> uppsala suite had some of the nicer tools for resetting B within pdb >> files, without having to do it manually (ugh - not appealing) - or >> writing an adhoc awk script (a very easy alternative, if you're >> familiar enough). >> >> Mark Robien >> >> >> On 3/1/2011 10:32 AM, Judith Reeks wrote: >>> Dear All, >>> >>> I am currently refining a structure using the latest experimental >>> version of Refmac (5.6) and there seems to be a problem with my Fo-Fc >>> map. There is a region where I have fitted residues to the electron >>> density but after refinement there is still positive electron density >>> assigned to the region despite the fact that residues fit the >>> electron density (see link below for a screenshot). Multiple rounds >>> of refinement have yet to get rid of this problem. I have checked the >>> PDB file and there does not appear to be any problems with this >>> region. Has anybody seen something like this before? >>> >>> http://i1083.photobucket.com/albums/j382/jreeks/Screenshot2011-03-01at1613402.png >>> >>> >>> Regards, >>> >>> Judith Reeks >>> >>> [log in to unmask] >>> >>> School of Chemistry >>> >>> University of St Andrews >>> >> >