5 Feb
2016
5 Feb
'16
8:25 a.m.
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