[phenixbb] question on molecular replacement

Andrew Purkiss-Trew Andrew.Purkiss-Trew at cancer.org.uk
Mon Jun 11 06:38:28 PDT 2012

For this resolution and type of problem, generating a Selenomethionine derivative is probably overkill.

Firstly, you can try abinitio methods such as Archimboldo (http://chango.ibmb.csic.es/ARCIMBOLDO/), which can work on 2Angstrom data given a helical structure.

Secondly, try generating derivative datasets. So, for example, Potassium Iodide may bind to the helices and give good anomalous phase information even on a home source. SIRAS can then be used to solve the structure. You will need to spend some time getting a good derivative, but it is generally quicker and cheaper than producing SeMet protein.

Good Luck,

Andrew Purkiss.
X-ray Lab Manager
London Research Institute.

From: phenixbb-bounces at phenix-online.org [phenixbb-bounces at phenix-online.org] On Behalf Of Machius, Mischa Christian [mischa_machius at med.unc.edu]
Sent: 11 June 2012 14:19
To: PHENIX user mailing list
Subject: Re: [phenixbb] question on molecular replacement

Besides what others said about the technical aspects of your molecular replacement endeavors:

Gel filtration is not the most accurate method to determine oligomeric status. Also, the content of the asymmetric unit may or may not have anything to do with the oligomeric status of a protein. So, best not to pay too much attention to that. First, try to find four copies, then another one, if there is still space and there is electron density indicating another molecule.

More importantly, designed structures often deviate significantly enough from 'real structures that molecular replacement becomes difficult or impossible. There is great satisfaction to be derived from a designed molecule being suitable as a search model in molecular replacement, but in the end you'd really want an independently determined structure. I assume that part of the motivation behind determining the crystal structure of your protein is to verify your design. If so, a completely independent structure would be desirable and would also convince any reviewer. Thus, I would recommend generating the seleno-methionine variant of your design (provided it contains any methionines) and solving it experimentally.

Best of luck!

On Jun 10, 2012, at 10:57 PM, <fn1 at rice.edu>

> Hi everyone,
> I have a dataset in P21221. The cell content analysis shows that there should be 5 monomers in asu. The gel filtration profile of this protein shows that it should form a tetramer.
> I have a simulated structure as searching model. I try to use the whole model or backbone model, and different resolution cutoff. When the whole model is used, the llg of solution is negative. When the backbone model is used, the llg is about 160 when searching for 4 monomers and tfz is 7.4. But the following autobuild process could not lower rfactor than 50%.
> I notice online that it might be useful to run self rotation function first to find ncs operators before doing mr. Is this true and how can i do this?
> Thank you in advance!
> Fengyun
> _______________________________________________
> phenixbb mailing list
> phenixbb at phenix-online.org
> http://phenix-online.org/mailman/listinfo/phenixbb

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