2KKZ
Solution NMR structure of the monomeric W187R mutant of A/Udorn NS1 effector domain. Northeast Structural Genomics target OR8C[W187R].
SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | 2D 1H-15N HSQC | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 2 | 2D 1H-15N HSQC | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 3 | 3D HNCO | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 4 | 3D HNCA | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 5 | 3D HN(CO)CA | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 6 | 3D HN(CA)CO | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 7 | 3D CBCA(CO)NH | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 8 | 3D HNCACB | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 9 | 3D simultaneous CN NOESY | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 10 | 3D 1H-13C NOESY aromatic | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 11 | 3D HCCH-COSY | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 12 | 2D 1H-13C HSQC high resolution (L/V methyl stereoassignment) | 1.2 mM [U-5% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 13 | 2D 1H-15N hetNOE | 1.2 mM [U-5% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 14 | 1D 1H-15N T1 and T2 | 1.2 mM [U-5% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 15 | 3D HCCH-TOCSY | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| 16 | 3D CCH-TOCSY | 0.71 mM [U-100% 13C; U-100% 15N] OR8w187r, 20 mM sodium phosphate, 100 mM sodium chloride, 50 mM arginine, 50 mM glutamic acid, 1 % glycerol, 50 uM DSS | 95% H2O/5% D2O | 0.1 | 6.9 | ambient | 300 | |
| NMR Spectrometer Information | |||
|---|---|---|---|
| Spectrometer | Manufacturer | Model | Field Strength |
| 1 | Bruker | AVANCE | 800 |
| 2 | Bruker | AVANCE | 600 |
| NMR Refinement | ||
|---|---|---|
| Method | Details | Software |
| simulated annealing | THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 2251 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS AND 203 DIHEDRAL ANGLE CONSTRAINTS; 0 HYDROGEN BOND CONSTRAINTS (18.5 CONSTRAINTS PER RESIDUE, 5.5 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 84 TO 217 BY PSVS 1.3). IN ADDITION, 75 RESOLVED N-H RESIDUAL DIPOLAR COUPLINGS FOR ORDERED RESIDUES WERE INCLUDED IN ALL STRUCTURE CALCULATIONS. STRUCTURE DETERMINATION WAS PERFORMED ITERATIVELY USING CYANA 3.0. THE 20 STRUCTURES OUT OF 100 WITH THE LOWEST TARGET FUNCTION WERE FURTHER REFINED BY RESTRAINED MOLECULAR DYNAMICS/ENERGY MINIMIZATION IN EXPLICIT WATER (CNS) WITH PARAM19 AND USING A NEUTRAL HISTIDINE TAUTOMER (ND1H FORM) AT RESIDUE 169. | TopSpin |
| NMR Ensemble Information | |
|---|---|
| Conformer Selection Criteria | structures with the lowest energy |
| Conformers Calculated Total Number | 100 |
| Conformers Submitted Total Number | 20 |
| Representative Model | 1 (lowest energy) |
| Additional NMR Experimental Information | |
|---|---|
| Details | THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY, STATIC LIGHT SCATTERING AND 15N T1/T2 RELAXATION. 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 WERE DETERMINED USING CYANA 3.0. BACKBONE (PHI/PSI) DIHEDRAL ANGLE CONSTRAINTS WERE OBTAINED FROM TALOSplus. ROTAMER STATES OF SPECIFIC ORDERED RESIDUES WERE CONSTRAINED IN THE FINAL STAGE OF THE STRUCTURE REFINEMENT BASED ON PROCHECK. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL HHHHHH): BACKBONE, 98.3%, SIDE CHAIN, 95.9%, AROMATICS, 97.8%, STEREOSPECIFIC METHYL, 100%, STEREOSPECIFIC SIDE CHAIN NH2: 80%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 84 TO 217, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 87-163,169-202: (A) RMSD (ORDERED RESIDUES): BB, 0.5, HEAVY ATOM, 0.9. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 88.4%, ADDITIONALLY ALLOWED, 11.6%, GENEROUSLY ALLOWED, 0.0%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.43/-1.38, ALL, -0.30/-1.77. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 17.12/-1.41 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 84-217): RECALL, 0.968, PRECISION, 0.911, F-MEASURE, 0.939, DP-SCORE, 0.740. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 13. (G) AGREEMENT WITH RESIDUAL DIPOLAR COUPLINGS (20 MODELS): CORRELATION COEFFICIENT (R): 0.992 (0.001); Q RMS: 0.125 (0.008). THE C-TERMINAL HIS TAG RESIDUES OF THE PROTEIN (HHHHHH) WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION. COORDINATES FOR THE FOLLOWING RESIDUES ARE NOT WELL DETERMINED [S(PHI) + S(PSI) < 1.8]: 84-86,164-168,203-END. |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | collection | TopSpin | 2.1 | Bruker Biospin |
| 2 | data analysis | TopSpin | 2.1 | Bruker Biospin |
| 3 | data analysis | Sparky | 3.112 | Goddard |
| 4 | peak picking | Sparky | 3.112 | Goddard |
| 5 | processing | NMRPipe | 2.3 | Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax |
| 6 | peak picking | NMRPipe | 2.3 | Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax |
| 7 | chemical shift assignment | AutoAssign | 2.4.0 | Zimmerman, Moseley, Kulikowski and Montelione |
| 8 | structure solution | CYANA | 3.0 | Guntert, Mumenthaler and Wuthrich |
| 9 | refinement | CNS | 1.1 | Brunger, Adams, Clore, Gros, Nilges and Read |
| 10 | structure solution | AutoStructure | 2.2.1 | Huang, Tejero, Powers and Montelione |
| 11 | structure validation | PSVS | 1.3 | Bhattacharya and Montelione |
| 12 | structure quality analysis | MolProbity | 3.15 | Richardson |
| 13 | pdb processing | PdbStat | 5.1 | Tejero and Montelione |
| 14 | refinement | TALOS | plus | Cornilescu, Delaglio and Bax |
