[buster-discuss] Refine occupancy on two different polypeptide chains

Anna Suarez Larsson anna at xray.bmc.uu.se
Thu Feb 1 12:14:54 CET 2018

Dear Clemens and other Buster users,

On 29 Jan 2018, at 09:48, Clemens Vonrhein <vonrhein at globalphasing.com> wrote:

> Dear Anna
> On Fri, Jan 26, 2018 at 10:17:01AM +0100, Anna Suarez Larsson wrote:
>> I am dealing with a pentameric structure composed of two different
>> short (80 aa) polypeptide chains, A and B, that are 40%
>> identical. In the crystal packing it looks to be no preferred
>> orientation for the ring with respect to the two different
>> proteins. I would like to combine the two chains in the same
>> structure and refine with Buster to get the relative occupancy.
> Yes, that should be doable.


>> We know from gel electrophoresis of the two proteins coexpressed
>> that the relation is 3:2 (A to B) between them, both in crystals and
>> in solution. The crystals diffract to about 2.0 � resolution but the
>> spots are streaky. The R-values when refining with only one of the
>> polypeptide chains are much lower for A compared to B.
> Ok.
>> My attempt to do the refinement in Buster using first pdb2occ and then run with the command -Gelly failed with the comment:
>> Interpreting CONSTANT and FREE cards: 
>> *** ERROR found in interpreting atom specifier from CONSTANT card
>> *** ERROR specifier=FixOcc
>> *** ERROR does not match any atoms
>> *** ERROR *** found processing constant cards
> Yes: we never anticipated that the complete model consists of atoms
> that will get their occupancy refined - so the FixOcc set/group is
> actually empty.
>> How should I do to be able to proceed?
> What happens if you remove that line (NOTE BUSTER_CONSTANT OCC FixOcc)
> from your Gelly file?

I got a long list of errors in the sanity check about ‘contact between two ‘Protein’ type atoms from distinct residues’ so I added 'StopOnGellySanityCheckError=no’ to the command line to test. The refinement then pushed the chains apart and the R-values rised. As a control I tried to run each pentamer separately (without gelly) and then the refinement went ok. 

>> Is it possible to refine double occupancy on all the five
>> polypeptide chains or is it required that I keep some of them with
>> fixed occupancy?
> You should be able to do that, although using pdb2occ might not be the
> most elegant way: this tool is mainly intended to handle occupancy
> refinement situations with some small(ish) localised items (alternate
> conformation side-chains, partially occupied ligands, alternate loop
> conformations etc).

Do you have any suggestions where to find documentation of how to do it otherwise?

> Please let us know if you hit another issue.
> Kind regards
> Clemens & Andrew (for buster-develop)

Thank you for your answer!

Best regards,

Anna Larsson
Laboratory of Molecular Biophysics
Department of Cell and Molecular Biology 
Uppsala University
Box 596 
SE-75124 Uppsala, Sweden

Tel. +46-18-4714983
E-mail: anna.larsson at icm.uu.se

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