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Jean I'm not a chemist and this is a complete guess, but... Is the density for the remainder of the C-terminal peptide clear, or is it possible that this residue is cleaved during the formation of the gem-diol? -bob On 7/10/06, jean <[EMAIL PROTECTED]> wrote:
*** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** Dear all I have a problem with the ligand in my protein-ligand structure: it's a peptide analogue and one of the peptide bond N atoms shows a very strong negative peak (<-10sigma) in the Fobs-Fcalc map. I am nearing the end of refinement and everything looks fine apart from this one atom. Resolution = 43.0 - 2.18A, Rcryst = 19.3% Rfree = 24.6%. I've been using CNS and O. I added the ligand as follows: build with ProDrg, place with Arp/warp in CCP4i using default settings and then some manual torsioning in O. I was careful to keep the peptide bonds planar. The Fobs-Fcalc and map looks fine for the whole ligand except for this one N. It also fits the composite omit map nicely. The ligand B-factors are ~35 - which seems a little high perhaps but not enough to warrant what I'm seeing. (I think occupancy is probably not 100%, although I haven't tried refining occupancies). The B-factor of the N in question is 57 - the highest in the ligand. I've done 3 build-refine steps since adding the ligand and the negative density hasn't changed.. Admittedly, these were limited to minor adjustments and I didn't carry out more than15 minimisation steps per cycle, however it doesn't look like the problem will be solved by further refinement. There is no positive density that might indicate incorrect positioning of the atom. I asked the organic chemist who synthesised this ligand and he assures me there is no way the atom could be anything other than a N. My only other thought is perhaps this atom is different, in terms of electrons, from a typical peptide N? It's the peptide bond before the ligand C-terminus. Also the ligand is not a true peptide: the N of the previous peptide bond (to the N-terminal side of the problematic one) has been replaced by a C and the adjacent carbonyl group has formed a gem-diol at the active site zinc ion. The offending N and the gem-diol are separated by two CH2's and a carbonyl group. Can anyone shed some light? Or at least can anyone suggest something else I could try? Jean Watermeyer
