[buster-discuss] Restrained geometry
vonrhein at globalphasing.com
Wed Feb 7 15:56:35 CET 2018
On Mon, Feb 05, 2018 at 01:04:36PM +0100, xgrcri at ibmb.csic.es wrote:
> Dear Buster List,
> we are currently working with a very large low-resolution structure
> and using BUSTER/TNT for refinement and map calculation.
What constitutes "low-resolution" in your case? Different people/labs
have a different idea about this ... 3A might be "high" for some and
very "low" for others ;-)
What can also be useful to know:
* Do you have non-crystallographic symmetry (NCS) within your
structure, i.e. multiple identical molecules within the asymmetric
* Was your structure determined by experimental phasing, molecular
replacement or simply re-using an existing (isomorphous) known
How homologous to your own structure/sequence are your starting
models (if used)? And at what resolution were those refined (in
comparison to your own low-resolution structure)?
> Given the limitation of the resolution, we would be very interested
> in applying enhanced rotamer, geometric (for bond lengths and
> angles) and Ramachandran restraints,
Good idea - as long as you are aware that e.g. imposing Ramachandran
restraints will invalidate the Ramachandran plot/statistics as a
validation tool. BUSTER currently doesn't provide this functionality.
> as the evaluation with MOLPROBITY after each refinement cycle gives
> a very high number of outliers
Are these large numbers in absolute terms or also as a percentage of
the total? If you have 50 outliers for 500 residues it would be
worrying - but if you have 5000 residues it seems less severe.
> despite a good overall Clashscore (6.14; 100%) and Molprobity Score
> (2.63; 96%).
A high percentile among structures of a similar (hence low) resolution
is not a convincing quality metric of course. Different Molprobity
versions also return slightly different clashscores.
Obviously, the goal is not necessarily to refine a model against the
Molprobity statistics, but rather against the X-Ray data ;-)
Have you looked at the Ramachandran and/or rotamer outliers? There are
in principle two situations:
* These residues have clear density that forces them to be an
"outlier". In that case you could try and check if e.g. a slightly
different rotamer also fits: at lowish resolution, a lot of
side-chains can easily be flipped around (ILE, LEU, VAL etc).
* These residues have no density and they get pushed around by other
forces (e.g. bad contacts) without any X-Ray term holding them in
place. You could consider truncating them down to Cbeta or removing
whole stretches completely.
Other points to consider:
* Have you tried (re)processing your data e.g. with autoPROC 
and/or using the STARANISO server  to check for anisotropy? If
your data has severe anisotropy that seems to sometimes help in
refinement and providing clearer density after BUSTER.
* If you started from a well-refined, high(er) resolution model it
can easily happen that refinement "looses" the good geometry
through refinement, because the X-Ray data is not providing the
additional information. In that case you could try so-called
LSSR targeting  to stay close to your good starting model.
* If you have NCS you should always use LSSR restraints via the
You could also use "-autoncs_noprune" so that all residues will be
within the NCS relation (no automatic pruning of significant
outliers) - at least at the beginning.
There is another flag (-sim_swap_equiv) that tries to re-introduce
NCS relations for symmetrical side-chains (ASP, GLU, PHE, TYR and
ARG) where a 180-degree rotation won't change the chemistry, but
only atom names. Such a change in atom names has a knock-on effect
when comparing NCS-related copies in your model.
This also means that you should always "Cancel" the suggestion by
Coot to "fix" nomenclature upon loading of your PDB file: one
cannot be sure that this fix will alter atom naming of all
NCS-related copies of the same residue in a consistent way and can
therefore lead to a break-down of NCS-relation ... which
"-sim_swap_equiv" can again try to fix, but it is better to not
introduce errors than to try and fix them later.
If you do manual rebuilding: make sure to do this in an NCS-aware
manner, eg. a rotamer change in one chain needs to be propagated to
all other chains (unless there is clear indication of differences
supported by density).
> Is there a way to impose such restraints?
Anything you can describe in terms of distances , angles  or
torsions  can be added to the restraints. We've done that for
RNA/DNA base-pairing or secondary-structure restraints, although this
is not (yet) in our distribution.
Finally, the latest release of BUSTER contains tools from the PDB_REDO
 suite to help with model rebuilding. This could be potentially
helpful in your case too .
Clemens, Claus & Gerard (for buster-develop)
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