[phenixbb] sequence independent model building possible?

[Kaushik Hatti]

Dear All,

Thanks for all the valuable inputs.
I was able to build side chains for the core of the protein.  Using this
sequence information, I was able to identify sequence family from Pfam.  It
seems like it has phosphate-binding domain (I could also see a blob which
looks like a phosphate).  I am using Multiple sequence alignment from the
closest Pfam hits to built sidechains for the rest of the protein.  The
latest scores are, R/Rfree: 28/37; LLG=3681; TFZ=39.2

I hope to solve it very soon.

Thanks,
Kaushik
P.S:
1) I had searched Dali too.  These hits (with less than 2 A RMSD), when
used as phasing model in Phaser failed to yield a solution.  The top hits
probably belong to the same fold but seems to have internal domain
movements.  However, the core was conserved (which I recognise now as
phosphate-binding domain).

2) Had also searched PDB for unit cell with similar dimensions without much
help.  Will checkout Nearest-Cell v2.0.  Thanks for the this.

3) ~20% of the structure is loop for which backbone isn't built.  I hope to
model this soon.
Thanks again for all the help.




On Sat, Feb 6, 2016 at 3:01 AM, Jon Agirre <jon.agirre at york.ac.uk> wrote:

> Dear Kaushik,
>
> if you're suspecting you've crystallised something else, perhaps you could
> try running your crystal parameters through the nearest-cell server (
> http://app.strubi.ox.ac.uk/nearest-cell/nearest-cell.cgi), which will
> scan the PDB for crystals that match the input one.
>
> Good luck,
>
> Jon
>
> On 5 February 2016 at 20:06, Christian Roth <christianroth034 at gmail.com>
> wrote:
>
>> Hi, besides the already excellent suggestions, you might want to try if
>> density modification (NCs, solvent flattening, histogram matching) improves
>> your map a bit further. If you can assign enough residues you improve your
>> maps than even further step by step. On top your stretches are than
>> definitely long enough for a blast search.
>>
>> Christian
>> On 5 Feb 2016 06:02, "Kaushik Hatti" <hskaushik at gmail.com> wrote:
>>
>>> Hello,
>>>
>>> Is abinitio model building possible for a map with poly alanine model at
>>> 1.9A resolution?
>>>
>>> We thought we had crystallised our protein of interest X, collected data
>>> at 1.9 A and all attempts to solve protein X (which has many homologs)
>>> through MR failed.  All attempts to re-crystallise the same protein also
>>> failed.
>>>
>>> Now, we think the initial protein which got crystallised could be a
>>> contaminant (we don't have any crystals left from this batch to check for
>>> the sequence of the crystallised protein).  Through various methods (and a
>>> bit of luck) we have arrived at a decent map with LLG : 3600 and TFZ: 22
>>> and R/Rfree : 37/41 (for a poly alanine model).
>>>
>>> I believe these scores indicate right fold.  As I still don't know the
>>> sequence information, is it possible to build sidechains directly from the
>>> map (I could only identify a couple of residues and the model largely
>>> remains PolyAla)?  Autobuild with Rebuild-in-place didn't help in
>>> identifying any more residues.
>>>
>>> I have also searched PDB database for similar structures. But, none of
>>> those are either from our expression system (E. coli) or organism of our
>>> protein of interest. Neither did I find any similar sequences from E. coli
>>> or our organism of interest.
>>>
>>> Any leads/suggestions would be helpful.
>>> Thanks,
>>> Kaushik,
>>> MRN Murthy lab,
>>> MBU,
>>> IISc, India
>>>
>>> --
>>> Stupidity is everyone’s birthright.  However, only the learned exercise
>>> it!
>>> --Kaushik (28Oct2014)
>>>
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>>
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>
>
>
> --
> Dr Jon Agirre
> York Structural Biology Laboratory / Department of Chemistry
> University of York, Heslington, YO10 5DD, York, England
> http://www.york.ac.uk/chemistry/research/ysbl/people/staff/jagirre/
> +44 (0) 1904 32 8253
>



-- 
Stupidity is everyone’s birthright.  However, only the learned exercise it!
--Kaushik (28Oct2014)
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