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unrefineable structure ?

Joe Krahn jkrahn at bilbo.bio.purdue.edu
Sat May 3 07:34:13 EST 1997

Dear All:

schiweck at argon.biophys.mpg.de wrote:
> Dear all,
> I am stuck with the problem of an "unrefineable" antibody (Fab fragment)
> structure. The situation is as follows.
> I have collected data to 3 Angstroems from several crystals. All data can
> easily processed with DENZO and give consistent results (primitive tetrago-
> nal spacegroup with dimensions of about 78, 78 and 143 Angstroems). In SCALE-
> PACK processing with Laue symmetry P41212 gives a Rmerge (about 6.9 %) which
> is only slightly higher than the one I get for Laue symmetry P41 (6.5 %).
> Pseudo-precession images from the data processed in P41 also indicate that the
> higher symmetric laue group is present. Assuming that either P41212 or the
> enantiomorphic spacegroup P43212 is correct, one gets a Matthews coefficent of
> 2.24 for one molecule in the asymmetric unit.
> Of course I then used the coordinates of this mol-rep solution for refinement
> in XPLOR. Rigid-body refinement in P43212 for the whole molecule or the
> individuals
> domains results in a drop from 48 to 42 % for the R-factor and from 47 to 44 %
> in Rfree. In the following refinement the R-factor drops to about 30 % whereas
> the Rfree first drop to about 41 % and then after the R-factor has reached a
> value of about 35 % rises again to a final value of 45 % ! This behaviour is
> independent of the refinement protocol. Even with torsion angle dynamics
> which should solve my problem of underdetermination, (99.3 % completeness to
> 3 A but still about 12000 parameters to refine with 9000 reflections for 10 to
> 3 A) I cannot refine my structure.

Considering the possibility of twinning, have you checked the cumulative
disribution of reflections, as listed by the CCP4 program TRUNCATE? 
This is a
valuable first hint at twinning, but also shows unusual distributions
for pseudo-
symmetry or anisotropic B's.  Of course, these last two cases will cause
refinement problems.  A recent structre (glmS I think) had 3 monomers
fit perfectly
to 3 of four orthorhombic AU's, with the fourth monomer shifted to
create a very
hard to find P2(1) actual space group.  I had a less strange case of
that gave very strange diffraction patterns, of C2 twinned with A2,
resulting in
impossible systematic absences (all three axes are approximately equal,
beta approximately 120 -- a real pain to index.)  Looking at the
Patterson map
made it possible to figure out what was going on.  ALWAYS LOOK AT THE
MAP, even if you don't expect to find anything.  It helps frequently.

It seems that you may have the correct structure, but are just refining
too many
parameters.  First of all, did you do any building in initial 2Fo-Fc
maps to get
a better starting point?  I didn't see this mentioned, but it is
certainly very
important if any major changes occured There could be some portion that
is just wrong.
Also, what does the average temp. factor seem to be? Perhaps there's a
lot of disorder.

I have a few suggestions for things that I have used in poorly
underdetermined cases
that worked out quite well.  First, try rigid body refinement of
multiple small
fragments.  I generally start with the whole molecule, and work towards
smaller fragments, down to fragments of every 5 residues.  This is still
far fewer
parameters than refining individual atoms.  I will also refine grouped
B's in the 5
residue fragments, starting not from a set Wilson B value, but from B's
of the original
structure which will definitely have a more accurate distribution for
most residues.

Another approach I once used was to tabulate all dihedral angles for 10
models from
3 different crystal structure, and apply these dihedral restraints to
the model during
refinement.  This will keep the whole structure similar to known
structures, but allow
considerable overall flexibility, and will apply stronger restraints to
that don't vary between structures.

Good luck,  Joe Krahn

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