Hi Dirk,
I disagree with your final sentence. Even if you don't apply NCS
restraints/constraints during refinement, there is a serious risk of NCS
"contaminating" your Rfree. Consider the limiting case in which the
"NCS" is produced simply by working in an artificially low symmetry
space-group (e.g. P1, when the true symmetry is P2): in this case,
putting one symmetry mate in the Rfree set, and one in the Rwork set
will guarantee that Rfree tracks Rwork. The same effect applies to a
large extent even if the NCS is not crystallographic.
Bottom line: thin shells are not a perfect solution, but if NCS is
present, choosing the free set randomly is *never* a better choice, and
almost always significantly worse. Together with multicopy refinement,
randomly chosen test sets were almost certainly a major contributor to
the spuriously good Rfree values associated with the retracted MsbA and
EmrE structures.
Best wishes,
Dean
Dirk Kostrewa wrote:
Dear CCP4ers,
I'm not convinced, that thin shells are sufficient: I think, in
principle, one should omit thick shells (greater than the diameter of
the G-function of the molecule/assembly that is used to describe
NCS-interactions in reciprocal space), and use the inner thin layer of
these thick shells, because only those should be completely independent
of any working set reflections. But this would be too "expensive" given
the low number of observed reflections that one usually has ...
However, if you don't apply NCS restraints/constraints, there is no need
for any such precautions.
Best regards,
Dirk.
Am 07.02.2008 um 16:35 schrieb Doug Ohlendorf:
It is important when using NCS that the Rfree reflections be selected is
distributed thin resolution shells. That way application of NCS should not
mix Rwork and Rfree sets. Normal random selection or Rfree + NCS
(especially 4x or higher) will drive Rfree down unfairly.
Doug Ohlendorf
-----Original Message-----
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Eleanor Dodson
Sent: Tuesday, February 05, 2008 3:38 AM
To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] an over refined structure
I agree that the difference in Rwork to Rfree is quite acceptable at
your resolution. You cannot/ should not use Rfactors as a criteria for
structure correctness.
As Ian points out - choosing a different Rfree set of reflections can
change Rfree a good deal.
certain NCS operators can relate reflections exactly making it hard to
get a truly independent Free R set, and there are other reasons to make
it a blunt edged tool.
The map is the best validator - are there blobs still not fitted? (maybe
side chains you have placed wrongly..) Are there many positive or
negative peaks in the difference map? How well does the NCS match the 2
molecules?
etc etc.
Eleanor
George M. Sheldrick wrote:
Dear Sun,
If we take Ian's formula for the ratio of R(free) to R(work) from his
paper Acta D56 (2000) 442-450 and make some reasonable approximations,
we can reformulate it as:
R(free)/R(work) = sqrt[(1+Q)/(1-Q)] with Q = 0.025pd^3(1-s)
where s is the fractional solvent content, d is the resolution, p is
the effective number of parameters refined per atom after allowing for
the restraints applied, d^3 means d cubed and sqrt means square root.
The difficult number to estimate is p. It would be 4 for an isotropic
refinement without any restraints. I guess that p=1.5 might be an
appropriate value for a typical protein refinement (giving an R-factor
ratio of about 1.4 for s=0.6 and d=2.8). In that case, your R-factor
ratio of 0.277/0.215 = 1.29 is well within the allowed range!
However it should be added that this formula is almost a
self-fulfilling prophesy. If we relax the geometric restraints we
increase p, which then leads to a larger 'allowed' R-factor ratio!
Best wishes, George
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-2582
*******************************************************
Dirk Kostrewa
Gene Center, A 5.07
Ludwig-Maximilians-University
Feodor-Lynen-Str. 25
81377 Munich
Germany
Phone: +49-89-2180-76845
Fax: +49-89-2180-76999
E-mail: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>
*******************************************************
--
Dean R. Madden, Ph.D.
Department of Biochemistry
Dartmouth Medical School
7200 Vail Building
Hanover, NH 03755-3844 USA
tel: +1 (603) 650-1164
fax: +1 (603) 650-1128
e-mail: [EMAIL PROTECTED]