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

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)

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)

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

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

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

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)

Structure determination Automated software: iteratively assign and remove bad assignments (CNS; ARIA) Iteration 0-7; 20 structures per iteration Last iteration; 100-200 structures Entire process is iterated (~nested) [~1 day over 20 desktop CPUs; ethernet, NFS; farm out calcs]

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

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)

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

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

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

Example: Map S2 on structure

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