Solution NMR structure of the C-terminal domain of the interferon alpha-inducible ISG15 protein from Homo sapiens. Northeast Structural Genomics target HR2873B
SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | 3D_15N-separated_NOESY | 0.81 mM U-13C,15N HR2873B, 50 mM ammonium citrate, 5 mM CaCl2, 1x protease inhibitor, 0.02% NaN3, pH 6.5 | 5% D2O / 95% H2O | 50 mM ammonium citrate, 5 mM CaCl2 | 6.5 | ambient | 293 | |
| 2 | 3D_13C-separated_NOESY | 0.81 mM U-13C,15N HR2873B, 50 mM ammonium citrate, 5 mM CaCl2, 1x protease inhibitor, 0.02% NaN3, pH 6.5 | 5% D2O / 95% H2O | 50 mM ammonium citrate, 5 mM CaCl2 | 6.5 | ambient | 293 | |
| 3 | HNHA | 0.81 mM U-13C,15N HR2873B, 50 mM ammonium citrate, 5 mM CaCl2, 1x protease inhibitor, 0.02% NaN3, pH 6.5 | 5% D2O / 95% H2O | 50 mM ammonium citrate, 5 mM CaCl2 | 6.5 | ambient | 293 | |
| 4 | high resolution 2D CH-HSQC (for stereospecific assignment of Val/Leu methyls) | 0.9 mM 5%-13C,U-15N HR2873B, 50 mM ammonium citrate, 5 mM CaCl2, 1x protease inhibitor, 0.02% NaN3, pH 6.5 | 5% D2O / 95% H2O | 50 mM ammonium citrate, 5 mM CaCl2 | 6.5 | ambient | 293 | |
| 5 | 2D 15N,1H heteronuclear NOE | 0.81 mM U-13C,15N HR2873B, 50 mM ammonium citrate, 5 mM CaCl2, 1x protease inhibitor, 0.02% NaN3, pH 6.5 | 5% D2O / 95% H2O | 50 mM ammonium citrate, 5 mM CaCl2 | 6.5 | ambient | 293 | |
| 6 | 3D TR backbone expts | 0.81 mM U-13C,15N HR2873B, 50 mM ammonium citrate, 5 mM CaCl2, 1x protease inhibitor, 0.02% NaN3, pH 6.5 | 5% D2O / 95% H2O | 50 mM ammonium citrate, 5 mM CaCl2 | 6.5 | ambient | 293 | |
| 7 | 3D HCCH-COSY, 3D HCCH-TOCSYs | 0.81 mM U-13C,15N HR2873B, 50 mM ammonium citrate, 5 mM CaCl2, 1x protease inhibitor, 0.02% NaN3, pH 6.5 | 5% D2O / 95% H2O | 50 mM ammonium citrate, 5 mM CaCl2 | 6.5 | ambient | 293 | |
| NMR Spectrometer Information | |||
|---|---|---|---|
| Spectrometer | Manufacturer | Model | Field Strength |
| 1 | Bruker | AVANCE | 600 |
| 2 | Varian | INOVA | 600 |
| NMR Refinement | ||
|---|---|---|
| Method | Details | Software |
| simulated annealing | THE STRUCTURES ARE BASED ON A TOTAL OF 976 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 80 DIHEDRAL ANGLE CONSTRAINTS, AND 40 HYDROGEN BOND CONSTRAINTS (14.6 CONSTRAINTS PER RESIDUE, 4.8 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 86 to 160 BY PSVS 1.3). STRUCTURE DETERMINATION WAS PERFORMED ITERATIVELY USING AUTOSTRUCTURE (XPLOR-NIH). AFTER A FINAL XPLOR CALCULATION USING THE CONSTRAINTS DERIVED FROM AUTOSTRUCTURE, THE 20 LOWEST ENERGY STRUCTURES OUT OF 100 WERE FURTHER REFINED BY RESTRAINED MOLECULAR DYANMICS/ENERGY MINIMIZATION IN EXPLICIT WATER (CNS). THE N-TERMINAL MET AND UNSTRUCTURED C-TERMINUS OF THE PROTEIN (LRGG + LEHHHHHH TAG) WERE INCLUDED IN ALL STRUCTURE CALCULATIONS BUT HAVE BEEN OMITTED FROM THIS DEPOSITION. | XwinNMR |
| NMR Ensemble Information | |
|---|---|
| Conformer Selection Criteria | structures with lowest conformational energy |
| Conformers Calculated Total Number | 100 |
| Conformers Submitted Total Number | 20 |
| Representative Model | 1 (lowest energy) |
| Additional NMR Experimental Information | |
|---|---|
| Details | THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. AUTOMATED BACKBONE ASSIGNMENTS WERE MADE USING AUTOASSIGN, AND THE SIDE CHAIN ASSIGNMENTS WERE COMPLETED MANUALLY. AUTOMATIC NOESY ASSIGNMENTS AS WELL AS DISTANCE AND HYDROGEN BOND CONSTRAINTS WERE DETERMINED USING AUTOSTRUCTURE. DIHEDRAL ANGLE CONSTRAINTS WERE DETERMINED USING HYPER. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING N-TERMINAL MET AND C-TERMINAL HHHHHH): BACKBONE, 99.8%, SIDE CHAIN, 95.3%, AROMATICS, 100%, STEREOSPECIFIC METHYL, 95.2%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 86 TO 160, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE 89-94,100-108,111-124,126-132,135-136,139-158: (A) RMSD (ORDERED RESIDUES): BB, 0.6, HEAVY ATOM, 1.1. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 86.7%, ADDITIONALLY ALLOWED, 12.7%, GENEROUSLY ALLOWED, 0.6%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.60/-2.05, ALL, -0.44/-2.60. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 28.41/-3.35. (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (ALL RESIDUES): RECALL, 0.978, PRECISION, 0.910, F-MEASURE, 0.943, DP-SCORE, 0.811. |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | collection | XwinNMR | 3.5pl6 | Bruker |
| 2 | collection | VNMR | 6.1C | Varian |
| 3 | data analysis | AutoAssign | 2.2.1 | Zimmerman, Moseley, Montelione |
| 4 | data analysis | Sparky | 3.110 | Goddard & Kneller |
| 5 | refinement | AutoStructure | 2.1.1 | Huang & Montelione |
| 6 | refinement | XPLOR-NIH | 2.11.2 | Clore et al |
| 7 | processing | NMRPipe | 2.3 | Delaglio et al |
| 8 | data analysis | PdbStat | 4.1 | Tejero & Montelione |
| 9 | data analysis | PSVS | 1.3 | Bhattacharya, Hang, Montelione |
| 10 | refinement | CNS | 1.1 | BRUNGER,ADAMS,CLORE,DELANO,GROS,GROSSE-KUNSTLEVE,JIANG,KUSZEWSKI,NILGES,PANNU,READ,RICE,SIMONSON,WARREN |
