Re: [ccp4bb] Molecular Replacement issues with a WD-40 7-bladed beta propeller

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Subject: Re: Molecular Replacement issues with a WD-40 7-bladed beta propeller
From: Matthew {- dot -} Franklin {- at -} IMCLONE {- dot -} COM Matthew {- dot -} Franklin {- at -} IMCLONE {- dot -} COM
Date: 2007-05-11

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From: Mark Agacan M {- dot -} Agacan {- at -} DUNDEE {- dot -} AC {- dot -} UK
Date: 2007-05-11

Subject: Re: Molecular Replacement issues with a WD-40 7-bladed beta propeller
From: Eleanor Dodson ccp4 {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2007-05-11

You dont say how close your sequence ID is for your new protein and the
model. It is often very hard to kick start refinement with low homology

And what about internal symmetry
Is there a Non crystallographic translation?
Does the self rotation function show a relationship between the two
molecules?
Does it show the 7 fold symetry?
Maybe you can verify your solutions fit with anv NCS. ( This is often
not easy!)
I use superpose molecules to a
) fit copy 1 to copy 2, and then
b) propellor blade 1 to 2 3 4 etc..

Then verify the rotation angles given are consistent with those in the
self rotation function.
MOLREP and POLARRFN both give a complete list of symmetry equivalents
for angles.

Then if the answer is right the struggle to refine begins..
Eleanor

Scott Coyle wrote:
> Hello,
> I'm an undergraduate and recently crystallized and obtained 2.9A
> diffraction data for a protein which is predicted to fold into a WD40
> 7-bladed beta-propeller structure (which has been crudely verified by
> cryo-EM by another lab). The space group appears to be I4(1) with unit
> cell parameters 118.936 118.936 85.456 90.000 90.000
> 90.000. Using a number of different search models (which I trimmed and
> aligned to my protein's sequence using Chainsaw) I have obtained a
> number of MR solutions placing 2 molecules in the AU with Phaser with
> high Z-scores (ranging from Z=9 to 12) that seem to pack together
> nicely, so I was hoping to use this technique to solve my structure.
> However, the initial Rfree for my best solution is relatively high
> (0.49) and all attempts to refine the structure result in the Rfree
> blowing up almost immediately. This makes me worry that the maps I'm
> generating may be too model-biased to use to generate a solution. I've
> tried using Prime and Switch to remove model bias but the resulting
> map looks worse than the starting map. As the predicted structure
> possesses so much radial symmetry (7-fold), I'm worried that my MR
> solutions will never be oriented correctly enough for me to be able to
> build a model. If anyone has any suggestions for tackling this kind of
> molecular replacement woe, I would greatly appreciate it. Otherwise I
> guess I'll just plan to collect experimental phasing information
> sometime in the near future.
>
> I'm not sure if this is the right place to be asking this question,
> perhaps you guys could direct me elsewhere.
>
> Thanks!
> -Scott
>
>

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Next message:
Subject: Filament Lifetime
From: Mark Agacan M {- dot -} Agacan {- at -} DUNDEE {- dot -} AC {- dot -} UK
Date: 2007-05-11