Dear CCP Community,
We have processed protein scattering data in space group C2221 to a resolution of 2.4 Å. The structure shows two protein molecules (chain A and B) in the asymmetric unit, related by a local two-fold symmetry axis. Initial rigid body refinement and subsequent full refinement of isotropic atomic B-factors (including all water and ligand molecules; using also non-crystallographic symmetry restraints) with the program REFMAC5 yielded R and Rfree values of 32.1and 33.9, respectively.
In the next step, after setting the B-factor to 30 Å^2, we carried out 6 cycles of TLS refinement followed by 10 cycles of isotropic atomic B-factor refinement (two TLS-bodies were defined: the two symmetry-related streptavidin chains A and B in the asymmetric unit). As expected, the R and Rfree values droped down by 1-2% to 29.6 and 32.5%, respectively. Checking the residual atomic B-factors their global average had changed from 13 Å^2 (without TLS refinement) to 2 Å^2, the lower B-factor boundary. In fact, all atoms in the .pdb file showed B-values of 2 Å^2. Interestingly, when we previously processed the same molecule in C2 we did not have this TLS problem.
Does anybody has an explanation why after TLS refinement, individual B-factor refinement is failing? Is this due to wrongly refined (i.e. too large) TLS parameters? Has this been observed before? Any suggestions to solve the problem?
Many Thanks, Christophe -- ************************************* Christophe Wirth Structural biology department Biozentrum, University of Basel Klingelberstrasse 70 CH-4056 BASEL *************************************
