[Xplor-nih] request for some help

[Michael Hodsdon]

***

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



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