Dear Joane,
Bert's recommendations are very good, but I'd like to add a little
caution. If just part of the molecule has bad density then it is not
unusual, but if the whole chain does not look very good (missing
side-chains or backbone) you may have a couple of things going on.
First, I would first suspect NCS issues (as Bert has) where you might be
trying to force too high a space group since an NCS operator might be
darn close to a crystallographic operator. Second it could also be
caused by twinning, but since your R-factors are pretty respectable I
would imagine that is probably not the case although you may want to
look into it. Third, it could be static disorder where the crappy
density might be two molecules on top of each other but one is slightly
shifted. A way to tell is to scale down in your electron density maps to
see if there are extra strands and helices next to the strands and
helices you have built. Hopefully this is not the case and it is just
NCS messing with you...
Jon
--
Jonathan P. Schuermann, Ph. D.
Beamline Scientist
NE-CAT, Building 436E
Advanced Photon Source (APS)
Argonne National Laboratory
9700 South Cass Avenue
Argonne, IL 60439
email: schue...@anl.gov
Tel: (630) 252-0682
Fax: (630) 252-0687
On 05/19/2011 10:06 AM, Van Den Berg, Bert wrote:
One more thing though: have you refined with the NCS restraints off or
on? Presumably on, seeing that you have a small gap between R and
Rfree? It may be worth turning the restraints off or modify the NCS
selections: if your 3 molecules are substantially different, applying
NCS may make things worse rather than better (ie you're averaging
things that are different).
Good luck, Bert
On 5/19/11 9:02 AM, "Joane Kathelen Rustiguel" <jkrustig...@usp.br> wrote:
Dear all
I am refining a structure at 3.4 A resolution that contains 3
molecules in the
a.u. The chain A sits on a 2-fold crystallographic axis forming
the dimeric
functional structure expected for this class of proteins. The
other two chains
B and C, which also form the functional dimer, seem to be,
somehow, a lot more
flexible than chain A. As a result, whereas the electron density
map, b-factor
and geometry for chain A is pretty reasonable for a 3.4 A resolution
structure, the refinement for the other two chains (B and C) does
not behave
well. Even playing with different weights for geometry, analysing
different
levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing
works. The map
for the helical regions is ok, but the electron density map for
strands and
loops of chains B and C are broken along the main chain, B-factors
are really
high, and the geometry keeps being distorted.
Right now, the R-factor and R-free are 24.2 and 28.6, respectively.
Any suggestions in how to proceed the refinement?
And even a more difficult question, how do we report this type of
structure?
How do we deposit those coordinates? We can certainly use chain A
as a model
to perform interesting studies of structure-function relationship,
but we know
that chain B and chain C have problems.
Any help will be greatly appreciated.
Regards
Joane
--
Joane Kathelen Rustiguel Bonalumi
Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP
Laboratório de Cristalografia de Proteínas
Departamento de Física e Química
Fone: +55.16.3602.4193
