1. Douglas Kojetin, Ph.D. UC College of Medicine
Using the CCHMC BMI cluster for NMR structure and dynamics calculations
Douglas Kojetin, Ph.D.
UC College of Medicine

2. Biomacromolecular Structure
Two principal methods (high-resolution)
X-ray crystallography
Solid-state
NMR spectroscopy
Solution-state*
Different from MRI; both use magnets
MRI: bulk density
NMR: atomic-level applications
Others (Small Angle X-ray Scattering; solution)

3. NMR Studies of …
3D structure determination (structure ensemble vs. static crystal representative)
NMR is limited (size, solubility)
Dynamics (time-scale movements)
Certain time-scale motions are important for protein function (interactions, catalysis, etc.)
Sample quality (folded? domains?)
Titrations/conditions (ligand, pH, temp)

4. Protein NMR Spectroscopy
ethanol
(O)H
(C)H3
(C)H2
1D 1H NMR Spectrum
ubiquitin

5. Protein NMR Spectroscopy
1D 1H NMR Spectrum
2D 1H-15N HSQC
Each “point” is sensitive to chemical environment
Ligands, temp, pH, etc.

6. Data Analysis: Step 1 Unambiguously assign all atoms in the protein
Backbone (HN, N, Ca, Cb, C’)
Side-chain
HNCACB, (2) HNCO/HN(CO)CA, (3) HNCA/HN(CO)CA

7. Structure determination
Start from an extended polypeptide chain
Constraints for structure calculation
through-space 1H-1H connectivities (NOEs)
Dihedral angles (predicted; chemical shifts vs. random coil database)
Hydrogen bonds
Residual dipolar couplings (orientation)

8. Structure determination
Automated software: iteratively assign and remove bad assignments (CNS; ARIA)
Iteration 0-7; 20 structures per iteration
Last iteration; structures
Entire process is iterated (~nested) [~1 day over 20 desktop CPUs; ethernet, NFS; farm out calcs]

9. Structure determination
*Solvent refinement
Fine tune NOE automated assignments
Better Ramachandran (stereochemical) quality; fewer atom clashes
AMBER: MPI capable, but calculations take ~3 hrs per structure; 100 separate, independent calculations
shell scripts to setup files, submit 100 jobs to queue; write working files to /tmp/XXX on node; copy important files to home dir; delete /tmp/XXX when finished)
Future MD calculations will take advantage of MPI

10. Dynamics calculations (time-scale motions)
Measure 15N backbone relaxation rates (measures motions on ps-ns time-scale)
Fit rates to motional models; use statistical model selection to choose best fit
Motional model; type of tensor describing system
Important motional parameters: S2 (order parameter) and Rex (ms time-scale motions; data cannot be described by ps-ns motions only)

11. Dynamics calculations (time-scale motions)
Nested, single calculations 1
1. Initial calculation
2. Analyze data vs. 4 tensor types: sphere, prolate, oblate, ellipsoid 2 2 2 2
3. Final selection of tensor;
monte carlo error estimation 3
4. Extract statistical information into
csv table
Repeated using different initial parameter estimates describing system
(NH bond length, corr. Timr; tensor params) 4

12. Dynamics calculations (time-scale motions)1
#PBS -S /bin/csh
#PBS -l walltime=24:00:00
#PBS -l nodes=1:ppn=1:opteron
... 2 2 2 2
#PBS -S /bin/csh
#PBS -l walltime=24:00:00
#PBS -l nodes=1:ppn=1:opteron
...
#PBS -W depend=afterok:CLUSTER_JOB x4 3 4

13. Dynamics calculations (time-scale motions)
#!/bin/sh
#1 QUEUE INITIAL RUN
local_tm=`qsub $JOB_DIR/local_tm`
local_tmid=`echo $local_tm | gawk -F '.' '{print $1}'`
sleep 2
#2a SETUP TENSOR RUNS
sed "s/CLUSTER_JOB/$local_tmid/g" $JOB_DIR/sphere \
> $JOB_DIR/sphere.mod
... prolate, oblate, ellipsoid (4 total)
#2b QURUE TENSOR RUNS
sphere=`qsub $JOB_DIR/sphere.mod`
sphereid=`echo $sphere | awk -F '.' '{print $1}’`
#3 QUEUE FINAL RUN
sed "s/FINAL_JOB/$sphereid:$prolateid:$oblateid:$ellipsoidid/g” \
$JOB_DIR/final > $JOB_DIR/final.mod
final=`qsub $JOB_DIR/final.mod`
finalid=`echo $final | awk -F '.' '{print $1}'`
#4 QUEUE FINAL STATS EXTRACTION
sed "s/STATS_JOB/$finalid/g" $JOB_DIR/stats \
> $JOB_DIR/stats.mod
stats=`qsub $JOB_DIR/stats.mod`
#PBS -S /bin/csh
#PBS -l walltime=24:00:00
#PBS -l nodes=1:ppn=1:opteron
...
#PBS -W depend=afterok:CLUSTER_JOB 1 2 2 2 2 3 4

14. Example: Map S2 on structure

15. Thanks!
CCHMC BMI
Prakash
Those who provide $$
Good Samaritan ER