2K5D
SOLUTION NMR STRUCTURE OF SAG0934 from Streptococcus agalactiae. NORTHEAST STRUCTURAL GENOMICS TARGET SaR32[1-108].
SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | 2D 1H-15N HSQC | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 2 | 2D 1H-13C HSQC | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 3 | 3D HNCO | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 4 | 3D HN(CA)CO | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 5 | 3D CBCA(CO)NH | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 6 | 3D HNCACB | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 7 | 3D HBHA(CO)NH | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 8 | 3D HCCH-COSY | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 9 | 3D HCCH-TOCSY aliphatic | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 10 | CCH-TOCSY aliphatic | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 11 | 3D simultaneous CN NOESY | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 12 | 3D 1H-13C NOESY aromatic | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 13 | 3D HNHA | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 95% H2O/5% D2O | 0.1 | 6.5 | ambient | 293 | |
| 14 | simultaneous CN NOESY | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 100% D2O | 0.1 | 6.5 | ambient | 293 | |
| 15 | 2D 1H-13C HSQC | 0.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 100% D2O | 0.1 | 6.5 | ambient | 293 | |
| 16 | 2D 1H-13C HSQC high res. (L/V stereoassignment) | 0.57 mM [U-5% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 100% D2O | 0.1 | 6.5 | ambient | 293 | |
| 17 | 2D 1H-15N hetNOE | 0.57 mM [U-5% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 100% D2O | 0.1 | 6.5 | ambient | 293 | |
| 18 | 1D 1H-15N T1 and T2 | 0.57 mM [U-5% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide | 100% D2O | 0.1 | 6.5 | ambient | 293 | |
| 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 1629 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 100 DIHEDRAL ANGLE CONSTRAINTS, AND 38 HYDROGEN BOND CONSTRAINTS (16.2 CONSTRAINTS PER RESIDUE, 5.0 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 110 BY PSVS 1.3). STRUCTURE DETERMINATION WAS PERFORMED ITERATIVELY USING CYANA 2.1. 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. | 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 2.1. BACKBONE (PHI/PSI) DIHEDRAL ANGLE CONSTRAINTS WERE OBTAINED FROM TALOS. HYDROGEN BOND CONSTRAINTS WERE DETERMINED USING BOTH AUTOSTRUCTURE AND CYANA, AND WERE APPLIED ONLY IN THE FINAL REFINEMENT STAGE (CNS) OF THE STRUCTURE DETERMINATION. 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, 99.5%, SIDE CHAIN, 97.2%, AROMATICS, 100%, STEREOSPECIFIC METHYL, 94.4%, STEREOSPECIFIC SIDE CHAIN NH2: 100%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 1 TO 110, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 18-50,53-67,70-72,74-78,81-83,99-108: (A) RMSD (ORDERED RESIDUES): BB, 0.7, HEAVY ATOM, 1.0. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 90.5%, ADDITIONALLY ALLOWED, 9.5%, GENEROUSLY ALLOWED, 0.0%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.55/-1.85, ALL, -0.28/-1.66. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 17.32/-1.45 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1-110): RECALL, 0.989, PRECISION, 0.916, F-MEASURE, 0.951, DP-SCORE, 0.751. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 15. 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]: 1-17,51-52,68-69,73,84-98,109-110. |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | collection | TopSpin | 2.0 | Bruker Biospin |
| 2 | chemical shift assignment | AutoAssign | 2.4.0 | Zimmerman, Moseley, Kulikowski and Montelione |
| 3 | processing | NMRPipe | 2.3 | Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax |
| 4 | peak picking | Sparky | 3.113 | Goddard |
| 5 | data analysis | Sparky | 3.113 | Goddard |
| 6 | structure solution | CYANA | 2.1 | Guntert, Mumenthaler and Wuthrich |
| 7 | structure solution | CNS | 1.2 | Brunger, Adams, Clore, Gros, Nilges and Read |
| 8 | refinement | CNS | 1.2 | Brunger, Adams, Clore, Gros, Nilges and Read |
| 9 | rpf validation | AutoStructure | 2.2.1 | Huang, Tejero, Powers and Montelione |
| 10 | structure validation | PSVS | 1.3 | Bhattacharya and Montelione |
| 11 | pdb analysis | PdbStat | 5.0 | Tejero and Montelione |
