First of all, I would like to thank you for your comments.
After consideration of all your comments, I conclude that there are
three possibilities.
1.) search for some particularly poorly-behaved regions using
parvati-server
a.) refining the occupancy of that atoms and/or
b.) tightening the restraints
Problems which have already been metioned:
If I tighten the restraints, the anisotropic model may not be
statistically justified, which seems to be the case.
Using all reflections may not help that much, because I chose a set of
1.5% for Rfree (~1300 reflections) to get as much data as possible for
the refinement. For my first tries of anisotropic refinement I used 5%
of the reflections for Rfree but the same problem arose, so that I
decided to cut the Rfree to 1.5%.
2.) Using shelxl
3.) TLS with multi-groups
Should be the safe way!?
I will try all the possiblities, but especially the tls refinement seems
to be a good option to be worthy to try.
Thanks for your helpful advices,
georg
Ethan Merritt schrieb:
On Tuesday 30 January 2007 04:11, Georg Zocher wrote:
I have a problem refining a structure against a 1.33 A resolution data
[snip]
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?
1.33A is very nice, of course, but it is not sufficient to guarantee that
anisotropic refinement will be well-behaved. There are two issues here.
One is the numerical stability of the refinement. The other is the worry
that the refined Uij values may not be very accurate even if the
refinement is well-behaved.
Numerical stability can be addressed by adjusting the restraint weights.
In refmac these are SPHE, RFAC, and RBON.
For some particularly poorly-behaved regions of the model you may
have to retreat to isotropic treatment of atoms.
The Parvati server may be of help here
http://skuld.bmsc.washington.edu/parvati/parvati.html
The other worry is that even after tightening up restraints to make
the refinement numerically stable, your fully anisotropic model may
not be statistically justified. I suggest that you compare the R/Rfree
values from full aniso refinement to those from multi-group TLS
refinement. We have found several high resolution structures
(1.2 - 1.5A) where a multi-group TLS model gives the same or better
R values than a tightly restrained full aniso model, yet has a
dramatically smaller number of thermal parameters.
The TLSMD server is here
http://skuld.bmsc.washington.edu/~tlsmd/
Ethan
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
