Hi Ethan, > > 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. > > I am virtually certain that refinement of individual anisotropic > U^ij terms cannot be justified at 1.8A. Too many parameters, > too few observations. In some cases (high solvent) there may be enough reflections to give your atoms anisotropic B-factors. Look for instance at PDB entry 1tv4, it has 19.5 reflections per atom, 1rzh has even more. When in doubt, I use a Hamilton test to see if anisotropic Bs are acceptable. That said, of course your residual B-factors after TLS can be anisotropic, but TLS + anisotropic Bs don't seem right to me either. You might as well use anisotropic B-factors and then extract TLS tensors from them. That was the original point of TLS anyway. Cheers, Robbie
> > > > > > > > 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 > > > > > > > > -- > Ethan A Merritt > Biomolecular Structure Center, K-428 Health Sciences Bldg > University of Washington, Seattle 98195-7742