A few comments that you might find useful:
1. yes, even if you don't apply NCS restraints/constraints there will
be correlations between
reflections in cases of NCS symmetry or pseudo-crystallographic NCS
symmetry.
2. Fabiola, Chapman, et al., published a very nice paper on the topic
in Acta D. 62, 227-238, 2006.
3. From my experience, the effects for low NCS symmetry are usually
small, except cases of
pseudo-symmetry which can be easily addressed by defining the test set
in the high-symmetry
setting. For high NCS symmetry, the effects are more significant, but
then the structure
is usually much better determined, anyway, due to averaging.
4. At least the first one of the mentioned MsbA and EmrE structures
had a very high Rfree in the
absence of multi-copy refinement ( ~ 45%)! So, the Rfree indicated
that there was a major
problem.
5. The Rfree should vary relatively little among test sets (see my
Acta D 49, 24-36, 1993 paper)
- if there are large variations for different test set choices then
the test set may be too small
or there may be systematic problems with some of the reflections causing
them to dominate the R factors (outliers at low resolution, for
example).
Axel Brunger
On Feb 7, 2008, at 9:57 AM, Dean Madden wrote:
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]
Axel T. Brunger
Investigator, Howard Hughes Medical Institute
Professor of Molecular and Cellular Physiology
Stanford University
Web: http://atb.slac.stanford.edu
Email: [EMAIL PROTECTED]
Phone: +1 650-736-1031
Fax: +1 650-745-1463
