<div dir="ltr"><div><div><div>Dear All,<br><br></div>Thanks for all the valuable inputs. <br></div>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<br><br></div><div>I hope to solve it very soon.<br><br></div><div>Thanks,<br></div><div>Kaushik<br></div><div>P.S: <br>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).<br><br></div><div>2) Had also searched PDB for unit cell with similar dimensions without much help. Will checkout Nearest-Cell v2.0. Thanks for the this.<br><br></div><div>3) ~20% of the structure is loop for which backbone isn't built. I hope to model this soon.<br></div><div>Thanks again for all the help.<br></div><div><br></div><div><br></div><div><br></div></div><div class="gmail_extra"><br><div class="gmail_quote">On Sat, Feb 6, 2016 at 3:01 AM, Jon Agirre <span dir="ltr"><<a href="mailto:jon.agirre@york.ac.uk" target="_blank">jon.agirre@york.ac.uk</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div dir="ltr">Dear Kaushik, <div><br></div><div>if you're suspecting you've crystallised something else, perhaps you could try running your crystal parameters through the nearest-cell server (<a href="http://app.strubi.ox.ac.uk/nearest-cell/nearest-cell.cgi" target="_blank">http://app.strubi.ox.ac.uk/nearest-cell/nearest-cell.cgi</a>), which will scan the PDB for crystals that match the input one. </div><div><br></div><div>Good luck,</div><div><br></div><div>Jon</div></div><div class="gmail_extra"><div><div class="h5"><br><div class="gmail_quote">On 5 February 2016 at 20:06, Christian Roth <span dir="ltr"><<a href="mailto:christianroth034@gmail.com" target="_blank">christianroth034@gmail.com</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><p dir="ltr">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.</p><span><font color="#888888">
<p dir="ltr">Christian</p>
</font></span><div class="gmail_quote"><div><div>On 5 Feb 2016 06:02, "Kaushik Hatti" <<a href="mailto:hskaushik@gmail.com" target="_blank">hskaushik@gmail.com</a>> wrote:<br type="attribution"></div></div><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div><div><div dir="ltr"><p dir="ltr">Hello,</p>
<p dir="ltr">Is abinitio model building possible for a map with poly alanine model at 1.9A resolution?</p>
<p dir="ltr">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. </p>
<p dir="ltr">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).</p>
<p dir="ltr">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.</p>
<p dir="ltr">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.<br></p>
<p dir="ltr">Any leads/suggestions would be helpful.<br>
Thanks, <br>
Kaushik,<br>
MRN Murthy lab,<br>
MBU,<br>
IISc, India</p>
<p dir="ltr">-- <br>
<font color="#999999">Stupidity is everyone’s birthright. However, only the learned exercise it!</font><br>
<font color="#999999">--Kaushik (28Oct2014)</font></p>
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</blockquote></div><br><br clear="all"><br>-- <br><div class="gmail_signature"><div dir="ltr"><font color="#999999">Stupidity is everyone’s birthright. However, only the learned exercise it!<br>--Kaushik (28Oct2014)</font></div></div>
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