Dear Kaushik,

at 1.9A resolution you might be able to improve the map contrast further by 
rebuilding the poly-Alanine model with shelxe. It removes model bias you may 
have in the data. That way it may be easier to identify side chains.

The program TurboFrodo has nice functionality to assign residue classes 
instead of residues, but if there is an alternative, you may not want to learn 
TurboFrodo.

If you crystal is large enough, you could send it for sequencing to identify 
it - that's probably the best way. I know that Kornelius Zeth has quite a lot 
of experience with this approach.

Regards,
Tim

On Friday, February 05, 2016 11:31:45 AM Kaushik Hatti 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)
-- 
--
Paul Scherrer Institut
Dr. Tim Gruene
- persoenlich -
OFLC/102
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A

You could try a couple of things: - running phenix.structure_search (from the command line only at the moment) on your current model - running autobuild without rebuild in place

On Feb 4, 2016, at 10:01 PM, Kaushik Hatti 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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