Solution NMR Structure of XF2673 from Xylella fastidiosa. Northeast Structural Genomics Consortium Target XfR39
SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | 2D 1H-15N HSQC | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 2 | 2D 1H-13C HSQC | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 3 | 2D 1H-13C HSQC_armatic | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 4 | 3D CBCA(CO)NH | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 5 | 3D HBHA(CO)NH | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 6 | 3D HNCACB | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 7 | 3D HCCH-TOCSY | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 8 | 3D HCCH-COSY | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 9 | 3D CCH-TOCSY | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 10 | 3D HNCO | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 11 | 3D 1H-15N NOESY | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 12 | 3D simutaeous NOESY | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 13 | 3D 1H-13C NOESY aromatic | 1.0 mM [U-100% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 6.5 | ambient | 293 | |
| 14 | 2D 1H-13C HSQC_high resolution | 1.0 mM [U-5% 13C; U-100% 15N] XfR39, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02 % NaN3, 50 uM DSS | 95% H2O/5% D2O | 100 | 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 STRUCTURES ARE BASED ON A TOTAL OF 1591 COMFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 100 DIHEDRAL ANGLE CONSTRAINTS, AND 42 HYDROGEN BOND CONSTRAINTS (18.8 CONSTRAINTS PER RESIDUE, 4.4 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 97 BY PSVS 1.3) STRUCTURE DETERMINATION WAS PERFORMED ITERATIVELY USING AUTOSTRUCTURE AND CYANA. THE 20 LOWEST ENERGY STRUCTURES OUT OF 100 WERE FURTHER REFINED BY RESTRAINED MOLECULAR DYNAMICS/ENERGY MINIMIZATION IN EXPLICIT WATER (CNS). THE C-TERMINAL HIS TAG RESIDUES OF THE PROTEIN (HHHHHH) WERE INCLUDED IN ALL STRUCTURE CALCULATIONS AND HAVE BEEN INCLUDED IN THIS DEPOSITION. COORDINATES FOR THE FOLLOWING RESIDUES ARE NOT WELL DETERMINED: 1-17, 67-68, 88-97. 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, DIHEDRAL ANGLE (TALOS) AND HYDROGEN BOND CONSTRAINTS WERE DETERMINED USING CYANA. COMPLETENESS OF NMR ASSIGNMENT: BACKBONE, 91.81%, SIDE CHAIN, 80.87%, AROMATICS, 60.00%, STEREOSPECIFIC METHYL, 82.35%, FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUE 1-97 PSVS 1.3), WHERE ORDERED RESIDUE RANGES COMPRISE: 18-66, 69-87. (A) RMSD (ORDERED RESIDUES): BB 0.82, HEAVY ATOM: 1.46 (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 92.8%, ADDITIONALLY ALLOWED: 7.2%, GENEROUSLY ALLOWED : 0.0%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z): PHI-PSI, -0.30/-0.87, ALL , -0.26/-1.54. (D) MOLPROBITY CLASH SCORE (RAW/Z): 16.58/-1.32. (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUE 1-97): RECALL, 1, PRECISION, 0.979, F-MEASURE, 0.99, DP-SCORE, 0.823. | 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) |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | collection | TopSpin | 2.1 | Bruker Biospin |
| 2 | chemical shift assignment | AutoAssign | 2.2.1 | Zimmerman, Moseley, Kulikowski and Montelione |
| 3 | peak picking | Sparky | 3.110 | Goddard |
| 4 | data analysis | Sparky | 3.110 | Goddard |
| 5 | structure solution | AutoStructure | 2.2.1 | Huang, Tejero, Powers and Montelione |
| 6 | structure solution | CYANA | 2.1 | Guntert, Mumenthaler and Wuthrich |
| 7 | processing | NMRPipe | 2.3 | Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax |
| 8 | refinement | CNS | 1.1 | Brunger, Adams, Clore, Gros, Nilges and Read |
| 9 | data analysis | PSVS | 1.3 | Bhattacharya and Montelione |
| 10 | pdbanalysis | PdbStat | 5.0 | Tejero and Montelione |
