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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has been replaced by a C and the adjacent carbonyl group has formed a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
