2HPU
Solution NMR structure of the apo-NosL protein from Achromobacter cycloclastes
SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | HNCA | 1.0 mM NosL, U-15N, 13C; 100mM sodium phosphate; 1mM EDTA; 1mM DTT; 90% H2O, 10% D2O | 90% H2O/10% D2O | 100mM sodium phosphate | 6.5 | ambient | 303 | |
| 2 | HNCO | 1.0 mM NosL, U-15N, 13C; 100mM sodium phosphate; 1mM EDTA; 1mM DTT; 90% H2O, 10% D2O | 90% H2O/10% D2O | 100mM sodium phosphate | 6.5 | ambient | 303 | |
| 3 | HNCACB | 1.0 mM NosL, U-15N, 13C; 100mM sodium phosphate; 1mM EDTA; 1mM DTT; 90% H2O, 10% D2O | 90% H2O/10% D2O | 100mM sodium phosphate | 6.5 | ambient | 303 | |
| 4 | CBCA(CO)NH | 1.0 mM NosL, U-15N, 13C; 100mM sodium phosphate; 1mM EDTA; 1mM DTT; 90% H2O, 10% D2O | 90% H2O/10% D2O | 100mM sodium phosphate | 6.5 | ambient | 303 | |
| 5 | C(CO)NH | 1.0 mM NosL, U-15N, 13C; 100mM sodium phosphate; 1mM EDTA; 1mM DTT; 90% H2O, 10% D2O | 90% H2O/10% D2O | 100mM sodium phosphate | 6.5 | ambient | 303 | |
| 6 | HCC(CO)NH | 1.0 mM NosL, U-15N, 13C; 100mM sodium phosphate; 1mM EDTA; 1mM DTT; 90% H2O, 10% D2O | 90% H2O/10% D2O | 100mM sodium phosphate | 6.5 | ambient | 303 | |
| 7 | 1H-13C-CT HSQC | 1.0 mM NosL, U-15N, 13C; 100mM sodium phosphate; 1mM EDTA; 1mM DTT; > 95% D2O | > 95% D2O | 100mM sodium phosphate | 6.5 | ambient | 303 | |
| 8 | HCCH-TOCSY | 1.0 mM NosL, U-15N, 13C; 100mM sodium phosphate; 1mM EDTA; 1mM DTT; > 95% D2O | > 95% D2O | 100mM sodium phosphate | 6.5 | ambient | 303 | |
| 9 | 3D 15N NOESY | 1.0 mM NosL, U-15N; 100mM sodium phosphate; 1mM EDTA; 1mM DTT; 95% H2O, 5% D2O | 95% H2O/5% D2O | 100mM sodium phosphate | 6.5 | ambient | 303 | |
| 10 | 15N NOESY-HSQC | 1.0 mM NosL, U-15N; 100mM sodium phosphate; 1mM EDTA; 1mM DTT; 95% H2O, 5% D2O | 95% H2O/5% D2O | 100mM sodium phosphate | 6.5 | ambient | 303 | |
| 11 | 13C NOESY-HSQC | 1.0 mM NosL, U-15N, 13C; 100mM sodium phosphate; 1mM EDTA; 1mM DTT; > 95% D2O | > 95% D2O | 100mM sodium phosphate | 6.5 | ambient | 303 | |
| NMR Spectrometer Information | |||
|---|---|---|---|
| Spectrometer | Manufacturer | Model | Field Strength |
| 1 | Bruker | DRX | 600 |
| 2 | Varian | INOVA | 800 |
| NMR Refinement | ||
|---|---|---|
| Method | Details | Software |
| Ambiguous Restraints for Iterative Assignment (ARIA 1.2),torsion angle dynamics, simulated annealing | XwinNMR | |
| NMR Ensemble Information | |
|---|---|
| Conformer Selection Criteria | structures with acceptable covalent geometry,structures with favorable non-bond energy,structures with the least restraint violations,structures with the lowest energy |
| Conformers Calculated Total Number | 100 |
| Conformers Submitted Total Number | 20 |
| Representative Model | 1 (closest to the average) |
| Additional NMR Experimental Information | |
|---|---|
| Details | Sequential and intra-residue 1H, 15N, and 13C backbone and side-chain chemical shift assignments were extracted from a series of double and triple resonance NMR experiments (HNCA, HNCO, HNCACB, CBCA(CO)NH, C(CO)NH, HCC(CO)NH, 1H-13C-CT HSQC,and HCCH-TOCSY) conducted on a DRX600. nOe data were obtained from 3D 15N NOESY experiments with mixing times (tmix) of 100, 120, and 140 msec and from 3D HCHC-NOESY (tmix=140 msec) spectra acquired at 600 MHz. Additional 15N NOESY-HSQC and 13C NOESY-HSQC experiments (nOe mixing times of 120 msec) were acquired on a Varian 800 INOVA (800 MHz) instrument. |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | processing | XwinNMR | 3.1 | Bruker Inc. |
| 2 | processing | NMRPipe | 2.4 | Delaglio |
| 3 | data analysis | NMRView | 5.0 | Johnson |
| 4 | structure solution | ARIA | 1.2 | Nilges |
| 5 | refinement | CNS | 1.1 | BRUNGER,ADAMS,CLORE,DELANO,GROS,GROSSE-KUNSTLEVE,JIANG,KUSZEWSKI,NILGES,PANNU,READ,RICE,SIMONSON,WARREN |
