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
>
>
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