Hi all -

I seem to be encountering a problem with Refmac refinement of a
structure containing anisotropic B factors. I have been using TLS
refinement and individual anisotropic B refinement, which may not be
correct technique, but it does lower my R and Rfree versus aniso B
refinement alone (see below). I am asking Refmac to output the TLS
contribution to the ANISOU lines, presumably in addition to the aniso B
refinement contribution. I also reset the temperature factors to 20 at
the beginning of each refinement round. The refinement resolution is 75
to 1.8 A, and the space group is C2, if it matters.

The problem comes when I take the output from such a run, model build in
Coot, and feed it back in for the next round of refinement. As you can
see from the summary table below (trimmed for brevity), the refinement
blows up rather badly, making the R factors and the geometry
substantially worse.

     Ncyc Rfact Rfree FOM -LL -LLfree rmsBOND zBOND
rmsANGL zANGL rmsCHIRAL $$
$$
        0 0.3376 0.3576 0.556 115671. 6318.1 0.0
0.0 0.0 0.0 0.0
        5 0.2676 0.2921 0.680 109411. 6009.2 0.0
0.0 0.0 0.0 0.0
        9 0.2529 0.2785 0.703 108589. 5978.4 0.0
0.0 0.0 0.0 0.0
       10 0.2516 0.2787 0.710 108459. 5975.2 0.0170
0.829 1.534 0.731 0.097
       15 0.2503 0.2876 0.711 108711. 6042.1 0.0241
0.998 1.729 0.833 0.111
       20 0.2739 0.3152 0.672 110787. 6147.3 0.0268
1.103 1.837 0.885 0.119
       25 0.2949 0.3388 0.638 112219. 6215.4 0.0255
1.049 1.808 0.866 0.116
       30 0.3114 0.3575 0.609 113094. 6257.7 0.0243
1.006 1.794 0.854 0.114

This refinement also throws out an enormous number of warnings about
adjacent atoms' B factors being substantially different. Most of these
warnings appear to involve the autobuilt riding hydrogens and their
adjacent heavy atoms.

If I use pdbcur to strip out the ANISOU lines, but otherwise keep the
file and refinement protocol unchanged, it goes along nicely:

     Ncyc Rfact Rfree FOM -LL -LLfree rmsBOND zBOND
rmsANGL zANGL rmsCHIRAL $$
$$
        0 0.2912 0.3233 0.667 112220. 6151.2 0.0
0.0 0.0 0.0 0.0
        5 0.2476 0.2787 0.751 107853. 5934.7 0.0
0.0 0.0 0.0 0.0
        9 0.2468 0.2764 0.754 107501. 5915.6 0.0
0.0 0.0 0.0 0.0
       10 0.2469 0.2763 0.754 107480. 5914.2 0.0170
0.829 1.534 0.731 0.097
       15 0.1933 0.2387 0.810 101501. 5723.3 0.0238
0.994 1.791 0.868 0.118
       20 0.1849 0.2327 0.817 100446. 5694.7 0.0239
0.992 1.826 0.884 0.120
       25 0.1818 0.2316 0.821 100034. 5681.0 0.0223
0.925 1.775 0.855 0.114
       30 0.1804 0.2296 0.824 99820. 5669.4 0.0221
0.913 1.763 0.848 0.113

(Without TLS refinement, the final R and Rfree would be 0.1896 and 0.2503.)

So, what's happening here? Does Refmac not like ANISOU lines in the
input PDB file? I don't usually work with structures at a resolution
high enough to warrant aniso B refinement, so I haven't encountered this
before.

Thanks for any advice,

Matt


--
Matthew Franklin, Ph. D.
Senior Research Scientist
New York Structural Biology Center
89 Convent Avenue, New York, NY 10027
(646) 275-7165