> On 08/22/11 03:12, intekhab alam wrote:
> Hi there
> I have a data of heterotrimer protein complex at 2.9A resolution.
> One protein consists of two domains. I tried phaser as well as molrep which gives a solution with only one domain.
> Rotation function and translation function were found to be fine for these solutions.
> I tried to find missing domain of the protein after fixing one of the domain or the other partner proteins using whole part or various truncations of missing domain.
> I also tried to find and build missing domain using Rosetta with the solutions of Molrep or Phaser as template.
> But, there were no solutions, or the solutions are clashed with other domain or proteins.
> Furthermore, R and Rfree is 30 and 40, respectively, and I could not reduce them further.
> There was almost no electron density map in the empty space so that I could not model manually.
>
> Plz guide me ,how can i look for the missing domain in the protein.
>
> regards
>
>
> --
> INTEKHAB ALAM
> LABORATORY OF STRUCTURAL BIOINFORMATICS
> KOREA UNIVERSITY, SEOUL
>
> Dear Intekhab,
>
> Before putting a lot of effort in trying to find the missing domain, you first should make sure it is there. It may be that only two proteins of the heterotrimer were packed in the crystal, or that one domain got clipped off during crystallization. The way to do it, is to run an sds page gel of a crystal, or a number of crystals if they are too small.
>
> Best regards,
> Herman
>
>
> As already noted, it is possible that the other domain is not present, or not ordered. Try refining a bit without it, and see what your stats look like.
>
> It is also possible that it is present, but minor clashes are causing good solutions to be rejected. Go into the output file and examine the TF scores before overlap rejection.
>
> The R and Rfree sound pretty good for an MR solution. About the failure of these numbers to come down, I'm not sure what you tried. Refinement with REFMAC5 should bring them down. You should see indications in the 2fo-fc and fo-fc maps that they are accurate - i.e. that there are real features showing up that are not in your model. You can test that also by deleting some prominent side chains.
>
>
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