On 22/02/2014 10:15, Mark van Raaij wrote:
As the excellent tips that you got indicate, lower R-factors can be
obtained by getting better data (better crystals, better data
collection, better data processing) or better fitting, i.e. refinement.
In this respect, I am impressed by the automatic data processing
protocols now being implemented. Also, the automatic local NCS
refinement in REFMAC seems very good for our recent structures.
But I would really want to make a general comment - not ALL structures
can be better than the average!
Except structures from the Lake Wobegon Center for Structural Biology,
of course.
There will always be structures with 5%
higher R/Rfree than the average in the same resolution range. Sometimes
this will be due to suboptimal refinement, but sometimes it may simply
not be possible to get better crystals and better data. Better not
necessarily in term of resolution, but in terms of disorders like you
describe for your plate-shaped crystals.
What I mean is that one should make all efforts to get better crystals
and data and refine structures as well as possible, but sometimes it may
not be possible to beat the average of the pdb and one should not get
too hung up by that. These structures should also be deposited and
published.
On the other hand, these "rules" that R-factor should be a certain value
at a certain resolution, may lead to suboptimal refinement. For example
the thought "my R-factor is already better than the average" could be
counterproductive and lead people to stop refinement prematurely.
Sometimes a structure will have Rs better than the average for the
resolution, but still better refinement could lower it further and this
should then be done. I can think of an MR solution using a very
homologous model that was refined at higher resolution, structures with
high NCS, or simply certain rock-solid proteins...
Another popular one is (was?) that Rfree should always be below 30%,
while several important structures justifiably have Rfrees quite a bit
higher (others perhaps have not been refined enough).
So while comparing R/Rfree to the average of existing structures is
useful, it may not necessarily be a sign that a structure is "bad" if
your Rs are 5 % higher, not should your Rs being at or below the average
be an excuse for stopping refinement too early.
Fear that ones Rs are not low enough may even lead to certain forms of
cheating, for example not keeping the Rfree reflections truly free.
On 22 Feb 2014, at 01:41, Chris Fage wrote:
Dear CCP4BB Users,
I recently collected a number of datasets from plate-shaped crystals
that diffracted to 1.9-2.0 angstroms and yielded very nice electron
density maps. There is no major density unaccounted for by the model;
however, I am unable to decrease Rwork and Rfree beyond ~0.25 and
~0.30, respectively. Probably due to the more 2-dimensional nature of
my crystals, there is a range of phi angles in which the reflections
are smeared, and I am wondering if the problem lies therein.
I would be grateful if anyone could provide advice for improving my
refinement statistics, as I was under the impression that the
R-factors should be ~5% lower for the given resolution.
A few more pieces of information:
-Space group = P21, with 2 monomers per asymmetric unit;
-Chi square = 1.0-1.5;
-Rmerge = 0.10-0.15;
-Data were processed in HKL2000 and refined in Refmac5 and/or
phenix.refine;
-PHENIX Xtriage does not detect twinning, but hints at possible weak
translational pseudosymmetry;
-I was previously able to grow one atypically thick crystal which
diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22.
Unfortunately, the completeness of the dataset was only ~90%.
Regards,
Chris
Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij
--
Andreas Förster
Crystallization and X-ray Facility Manager
Centre for Structural Biology
Imperial College London
