*** I have made multiple attempts to send this message to the list and I am not sure what I am doing wrong. So, if it ends up that I have sent a number of copies of the same message to everyone's inbox, I sincerely apologize.
*** Greetings All, I have previous experience using CYANA to calculate structures using NOE restraints. Now, I am trying to include RDCs and am migrating over to Xplor-NIH. I am having a few difficulties and was hoping to "beg" for some assistance/advice. We are working on a 150 residue protein. We have nearly complete (97%) assignments and have picked a large number of NOESY correlations from 3D NOESY-HSQC spectra. Using "NOEASSIGN" (previously known as CANDID) in CYANA, these NOEs were easily assigned in a fully automated manner resulting in a nicely converged ensemble of structures (backbone RMSD < 1 Angstrom). We next converted the ~3000 independent distance restraints from CYANA to Xplor-NIH format. This was no easy task given the very different ways that CYANA and Xplor-NIH handle non-stereospecifically assigned hydrogens. I am fairly certain that the conversion utility bundled in CYANA 2.1 does NOT convert the NOEs correctly. I ended up writing my own AWK script to do it. Has anyone else tried to do this? I am fairly confident that my conversion is correct as I was able to import the converted restraints into Xplor-NIH and test them against one of the fitted structures from CYANA (which also had to be converted!) and got agreement. Surprisingly, when I try to use Xplor-NIH to calculate ensembles of structures using these converted distance restraints, I CANNOT achieve as good convergence as I could in CYANA. Using a local Beowulf cluster I calculate 100 structures and keep the best 20-40. I always had 10 - 20 frequently violated NOEs and a LOT of VDWs violations (20 - 60). I am guessing this is a consequence of the difference in the potential (pseudo-energy) functions used for the two methods. For example, in Xplor-NIH I am using this "RAMA" term, which I don't think there is an equivalent in CYANA. I began to suspect that my distance restraints from CYANA were just a little to "tight" (and thus conflicting with other terms in the potential function). So, I went back and loosened them up a bit (with CYANA's calibration routine) and I got many fewer NOE violations, but still a lot of VDW violations. Still, I felt this was progress. At this point, I had finally measured RDCs in Pf1. I started by just throwing in the N-N RDCs along with the above described "looser" NOEs. I used one of the example refine.py scripts (attached) and I took one of the fitted structures from CYANA as the starting model. The calculated structures are in good agreement with the RDCs (which was very nice to see) with tensor values of approx -16 and 0.6 (Da and Rh). But, I still get more NOE and VDW violations than I was expecting. I have to keep the identity of this protein a little quiet, but I have attached the refine.py script and the ".xplorInput_##.sa.stats" output file. Overall, the structures look the same as those created by CYANA, but they aren't as precise and the geometry isn't as good. There must be something I am doing wrong and I would love for some to point it out for me. Many, many thanks to anyone who read this far. Mike -- Michael E Hodsdon, MD, PhD Assistant Professor of Laboratory Medicine and Pharmacology Yale University School of Medicine email: michael.hodsdon at yale.edu http://info.med.yale.edu/labmed/faculty/hodsdonm.html 203.737.2674 (office) 203.737.2684 (lab) 203.915.0780 (mobile) 203.688.8704 (fax) Administrative Assistant: Vilma Moreno phone: 203.737.5934; email: vilma.moreno at yale.edu U.S. Postal Address: P.O. Box 208035, Laboratory Medicine Yale University School of Medicine 333 Cedar Street New Haven, CT 06520-8035 Shipping Address: CB462b, Laboratory Medicine 200 South Frontage Road New Haven, CT 06520 -------------- next part -------------- An HTML attachment was scrubbed... URL: http://dcb.cit.nih.gov/pipermail/xplor-nih/attachments/20070318/86aa0df5/attachment-0001.html -------------- next part -------------- A non-text attachment was scrubbed... Name: refine.py Type: application/octet-stream Size: 11853 bytes Desc: not available Url : http://dcb.cit.nih.gov/pipermail/xplor-nih/attachments/20070318/86aa0df5/attachment-0002.obj -------------- next part -------------- A non-text attachment was scrubbed... Name: sa.stats Type: application/octet-stream Size: 21295 bytes Desc: not available Url : http://dcb.cit.nih.gov/pipermail/xplor-nih/attachments/20070318/86aa0df5/attachment-0003.obj
