[phenixbb] multi-domain protein data, and multi-component/full-featured Phaser-MR

[Emilia C. Arturo (Emily)]

Hello.

I have been reading the tutorials and the 2007 McCoy (Acta D) paper about
how to use the full-featured Phaser-MR tool (with the Phenix GUI) to do MR.
I am struggling with how to define the Ensembles, and the Search procedure.
Specifically, after setting up the Ensembles and the Search procedure, as
I'd deduced that they should be done, I ran the script and get the
following message:

>>Search request requires more scattering than defined in composition.
Composition increased to accommodate search components.<<

I guess that I have not understood how to set up the multi-component search
to do what I want for it to do: I'd wanted the tool to search for the best
placement of individual domains, given a set of diffraction data and a
collection/combination of pdb files that contains coordinates for
individual domains.

This is the context:

The diffraction data is from a crystal of a three-domain protein. The first
model I used for MR was a crystal structure of the largest of these
domains, which had been expressed and purified alone. With this one domain
as a search model (using the single-component Phaser-MR) I obtained a
solution without a problem, but now have density into which I will need to
place the other two domains.

The second-largest domain has also been crystallized (also on its own). Its
structure (as a monomer or dimer) is available from different organisms,
some dimers engage different inter-chain interfaces, and some have ligands
bound at different interfaces. There does exist a crystal structure for the
three-domain protein, and its biological assembly is 4. I have reason to
think that this new crystal is of this same protein in a different
configuration - that is, in this crystal structure the three domains are
oriented differently to one another than they were in the first 3-domain
structure. In solution this protein can be a dimer or tetramer.

The ASU for this dataset is 2 chains, and I assume that each chain has all
three domains intact.

This diffraction data is of relatively poor resolution (~3.8A).

So, I configured the Phaser-MR run as follows, using the AP2 and Beta-blip
case studies as examples:

- In the 'Ensembles' tab I created 17 different ensembles, each with a
different combination of single-domain structures (or with just the single
largest domain, as in the original MR single-component search).

- In the 'ASU contents' tab I provided the sequence of the full-length
(3-domain) protein, and set 'Number of copies' to 2.

- In the 'Search strategy' tab I added each of the 17 Ensembles as a
separate 'Component' with 'Copies to search for' set to 2. This seems
redundant to what I'd put in to the 'Ensembles' tab.

I thought that a 'component' meant a single pdb file representing a single
chain or domain (in this case) while an 'Ensemble' meant a collection of
components so that, e.g., a 3-domain chain could be described as an
ensemble of 3 components.

Do you have advice on how to set up this search correctly?

Regards,

Emily.



-- 
"Study as if you were going to live forever; live as if you were going to
die tomorrow." - Maria Mitchell

"I'm not afraid of storms for I'm learning to sail my ship."  - Louisa May
Alcott
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