Hi Jon
On 30 Oct 2010, at 00:46, Tom Huxford wrote:
Hi all,
I'm working with good quality relatively complete x-ray diffraction
data collected to a resolution limit 2.6 Å from a crystal of a protein
with a small molecule ligand bound. I ran MR from 10-4 Å and then did
maximum likelihood rigid body refinement in Refmac5 against data from
50-3.5 Å. Now I would like to run restrained refinement from 50-3 Å.
The reason for doing this is that, in order to minimize the divergence
between R-cryst and R-free during refinement my advisor who, by the
way, is forwarding this e-mail for me (and editing it so please don't
bash him too mercilessly) suggested I first build in the ligand and
newly positioned polypeptide loops and refine against data at a lower
resolution limit before opening it up to all the available data.
I personally would not first build in the ligand and refine against
low res data...
Assuming the ligand is what you're after, I would:
(a) leave the ligand out of the time being
(b) refine the model using all data
(c) build the ligand in
(d) refine the model using all data
I'm not clear why doing restrained refinement using limited data first
should help..
Apparently this has worked well for him in the past (and it has!).
The
problem is that I'm to the point where I'd like to extend the
resolution down to 3 Å during restrained refinement but even if I set
the range from 50-3 Å in the ccp4i window refinement only happens from
50-3.5 Å. If I take a step back and do the rigid body refinement from
50-3 Å and then carry out restrained refinement from 50-3 Å it works
fine. Why would the limits imposed by rigid body refinement cause the
subsequent restrained refinement to be stuck at the rigid body
refinement's resolution limits?
Are you using the original reflection file?
Best
Roberto
Thanks for any thoughts,
Jon Fleming
Graduate Student
Structural Biochemistry Laboratory (Huxford Lab)
Department of Chemistry & Biochemistry
San Diego State University
---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax +44 (0)20-7848-6435
e-mail roberto.stei...@kcl.ac.uk
