If you have used TLS the B factors listed for the residues are the
residual values AFTER the TLS is applied so they are correctly
restrained to be quite close - the differences in the molecules should
be expressed in the hard-to-visualise TLS parameters.
The first check is to run
TLSANL and ask to get out the isotropic B equivalent after applying a
TLS operator. This might show what you expect - that one residue is well
defined whilst others are very floppy.
I usually start TLS by setting all B factors to the Wilson B estimated
in TRUNCATE or somewhere, then follow the GUI protocol - 10 cycles of
TLS followed by 10 cycles of restrained refinement with NCS restraints.
Is that what you have done?
Eleanor
ANA MARIA MISIC wrote:
Hello - I have nearly completed a crystal structure at 2.6 A resolution. There
are 3 monomers per asymmetric unit (same protein in different conformations),
and I have been using non-crystallographic symmetry during the refinement
process (REFMAC5). I have been isotropically refining B-values and using TLS.
My problem is that the B-values appear to be too tight as a result of
non-crystallographic symmetry. For example, an Asp residue has well-defined
density, and atomic B factors of about 40. However, the adjacent lysine, with
no density for the side chain has B factors of 42. This does not make sense
for them to have nearly identical B values, when there is differing density.
This may be a consequence of having a residue with well defined density in
chain A and chain B, but not chain C. When plotting residue number vs. B
factor, the values seem to be too tightly grouped to make sense. Loosening NCS
restraints leads to more variability in B factors, a lower R factor,
but higher R-free.
My question is how do I keep NCS, and allow B-factors to vary more so I have
more reasonable values? I would appreciate any ideas you have. Thank you in
advance for your time and assistance.
Ana M. Misic
Research Assistant
Department of Biomolecular Chemistry
Department of Bacteriology
University of Wisconsin - Madison
420 Henry Mall, Rm 230
Madison, WI 53706
Lab Phone: (608)265-9282
Fax: (608)262-9865
