After reading Ian's message and the first followups:
I think it's really important to keep the free-R set through the
initial anisotropic refinements, especially at resolutions 1.3 - 1.5
A where anisotropic refinement may or may not be warranted. I've
seen several instances where R dropped a couple of percent and Rfree
hardly budged (0.2 - 0.5 percent), indicating that, even though the
resolution and data/parameter ratios may have looked promising,
anisotropic refinement wasn't warranted. (In several cases TLS
refinement with just a few groups resulted in a larger drop in Rfree
than full anisotropic refinement.)
I don't think any of the messages have actually advocated dropping
the free R reflections before anisotropic refinement, however just
because you have 1.x A (fill in your favorite thresh hold) resolution
data doesn't automatically mean anisotropic refinement is justified.
Sue
On Jan 30, 2007, at 10:34 AM, Ian Tickle wrote:
I believe the recommended procedure is to refine against all data
before
submission, using the same set of parameters and weights as for the
last
refinement against the working set alone, after all why throw away 5%
(or even 10% in some cases) of your hard-won data? The purpose of
Rfree
is 1) to check for gross errors in the model, i.e. Rfree should not be
significantly greater than (or indeed less than) its expected value
(which depends essentially on the observation/parameter ratio), and 2)
to check for over-parameterisation and/or incorrect relative weighting
of X-ray terms & geometry (i.e. Rfree should be minimal at convergence
of refinement at maximal overall likelihood for an appropriate
choice of
variable parameters and weights). Once your structure has passed
these
tests satisfactorily there's no further need for the test set.
Basically you do all your cross-validation before you abandon your
test
set.
There is however a difficulty with this procedure, and it may not be
practical to carry it out: namely ML refinement uses the test set
to get
unbiased estimates of the sigma-A's, so it may not actually be
formally
correct to refine without a test set using e.g. Refmac. Other
well-known refinement packages (e.g. Shel-X) don't have this problem.
-- Ian
-----Original Message-----
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On
Behalf Of David Briggs
Sent: 30 January 2007 16:10
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] problem with anisotropic refinement
using refmac - Sacrosanct R-free
"...They might
also get rectified when you use all of your data for
refinement, meaning
when you are not using any R free..."
Sorry to go off on a little tangent here - but doesn't
refining against ALL of your data generally mean that your
RFree is no longer your "free" and cannot be used for
cross-validation...
I was always taught that the reflections within the test set
are sacrosanct - is this not also the case for circa atomic
resolution data? Perhaps someone more learned than I could
shed a little light on this...
Dave
Hallo to all out there,
>
> I have a problem refining a structure against a 1.33
A resolution data
> set. Using REFMAC (version 5.2.0019, also tested
version 5.3) with
> isotropic B-factor refinement resulted in an valid
model with R /
> R(free) of 17.8 / 19.2%.
>
> To finish the model I tried to use anisotropic
refinement which should
> be possible/reasonable because of an observable to
parameter ratio of
> about 2.8. This refinement yielded to a much better R
/ R(free)-Factor
> of 14.1 / 16.8% and converged after about 15 cycles
of anisotropic
> refinement. However the problem is that running a few
cycles of
> refinement resulted in warnings, logged as "Problem
in MAKE_U_POSITIVE".
>
> So what would be the right solution to overcome this problem?
>
> Some further information about the data set:
>
> spacegroup P 6(5) 2 2, one protomer per ASU
> cell axis 76.615 76.615 209.787
90.00 90.00 120.00
> unique reflections 83156 (6220)
> Completeness 98.6 % (87.9%)
> I/Sigma 21.4 (3.5)
> Rmrgd-F 5.9% (35.0%)
>
> Maybe the refmac-script will be of some help (some
other BFAC restraints
> and SPHE/RBON parameter tested, the following example
takes care of
> reasonable distribution of anisotropy):
>
> #!/bin/bash
> refmac5 hklin ../gz_ccp4.mtz \
> hklout gz_aniso_01f.mtz \
> xyzin ./gz_iso.pdb \
> xyzout gz_aniso_01f.pdb \
> libin ../llp_citrat_fitted.cif \
> << end_ip > refmac.log
> LABI FP=F_cit_01 SIGFP=SIGF_cit_01 FREE=FreeR_flag
> LABO FC=FC FWT=FWT PHIC=PHIC PHWT=PHWT DELFWT=DELFWT
PHDELWT=PHDELWT
> FOM=FOM
> NCYC 20
> REFI TYPE RESTRAINED
> REFI RESI MLKF
> REFI METH CGMAT
> REFI RESO 25 1.33
> REFI BREF ANISOTROPIC
> SCAL TYPE BULK
> SCAL LSSC ANISO NCYCLES 10
> SCAL MLSC NCYCLES 10
> WEIG MATRIX 1.25
> SPHE 30.0
> RBON 30.0
> BFAC 0.5 2.0 4.0 4.0 6.0
> MAKE CHECK ALL
> MAKE HYDROGEN ALL
> MAKE HOUT NO
> MAKE PEPTIDE NO
> MAKE CISPEPTIDE NO
> MAKE SSBRIDGE NO
> MAKE CHAIN YES
> MAKE SYMMETRY YES
> MONI MANY TORS 10 DIST 10 ANGL 10 VAND 10 PLANE 10
CHIR 10 BFAC 10 BSPH
> 10 RBOND 10
> BINS 20
> PNAM gz
> DNAM gz
> USEC
> END
> end_ip
>
> The final refinement statistic:
>
> Resolution limits = 25.000 1.330
> Number of used reflections = 81889
> Percentage observed = 98.6122
> Percentage of free reflections = 1.5000
> Overall R factor = 0.1409
> Free R factor = 0.1681
> Overall weighted R factor = 0.1348
> Free weighted R factor = 0.1641
> Overall correlation coefficient = 0.9763
> Free correlation coefficient = 0.9688
> Overall figure of merit = 0.9183
> ML based su of positional parameters = 0.0274
> ML based su of thermal parameters = 1.5420
> rmsBOND = 0.014
> rmsANGLE = 1.569
>
> Thanks in advance,
> georg zocher
>
>
--
---------------------------------------
David Briggs, PhD.
Father & Crystallographer
www.dbriggs.talktalk.net
iChat AIM ID: DBassophile
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