2KLB
NMR Solution structure of a diflavin flavoprotein A3 from Nostoc sp. PCC 7120, Northeast Structural Genomics Consortium Target NsR431C
SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | 2D 1H-15N HSQC | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C | 90% H2O/10% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 2 | 2D 1H-13C HSQC | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C | 90% H2O/10% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 3 | 3D CBCA(CO)NH | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C | 90% H2O/10% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 4 | 3D HNCACB | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C | 90% H2O/10% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 5 | 3D HNCO | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C | 90% H2O/10% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 6 | 3D HNCA | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C | 90% H2O/10% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 7 | 3D HN(CO)CA | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C | 90% H2O/10% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 8 | 3D HBHA(CO)NH | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C | 90% H2O/10% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 9 | 3D 1H-13C NOESY | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C | 90% H2O/10% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 10 | 3D 1H-15N NOESY | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C | 90% H2O/10% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 11 | 2D 1H-15N HSQC | 1.18 mM [U-10% 13C; U-100% 15N] NsR431C | 100% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 12 | 2D 1H-13C HSQC | 1.18 mM [U-10% 13C; U-100% 15N] NsR431C | 100% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 13 | 2D 1H-15N HSQC | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C 100% D2O | 100% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 14 | 2D 1H-13C HSQC | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C 100% D2O | 100% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 15 | 3D 1H-13C NOESY | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C 100% D2O | 100% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| 16 | 3D HCCH-TOCSY | 1.18 mM [U-100% 13C; U-100% 15N] NsR431C | 90% H2O/10% D2O | 5mM CaCl2, 200mM NaCl | 6.5 | 1 atm | 293 | |
| NMR Spectrometer Information | |||
|---|---|---|---|
| Spectrometer | Manufacturer | Model | Field Strength |
| 1 | Bruker | AVANCE | 600 |
| 2 | Bruker | AVANCE | 800 |
| NMR Refinement | ||
|---|---|---|
| Method | Details | Software |
| simulated annealing | 140 structures were calculated and 20 best conformers were refined in a shell of water using CNS. Initial dihedral angles were obtained using TALOS. Final quality scores were determined using PSVS software. Ordered residues are defined as: 2 - 9, 12 - 27, 31 - 36, 40 - 57, 61 - 79, 84 - 85, 87 - 89, 96 - 108, 110 - 111, 122 - 146; (a) RMSD (ordered residues) all Backbone atoms: 1.0A; all heavy atoms: 1.4A; (b) Ramachandran statistics for all ordered residues: Most favoured: 92.1%; Additionally allowed: 7.2%; Generously allowed: 0.6%; disallowed: 0.2%; (c) Procheck scores for ordered residues (Raw/Z)phi-psi -0.04/0.16, All:-0.10/-0.59. (d) MolProbity clash score (Raw/Z) 22.23/-2.12. | AutoAssign |
| NMR Ensemble Information | |
|---|---|
| Conformer Selection Criteria | structures with the lowest energy |
| Conformers Calculated Total Number | 140 |
| Conformers Submitted Total Number | 20 |
| Representative Model | 1 (lowest energy) |
| Additional NMR Experimental Information | |
|---|---|
| Details | Data acquisition on B600 was performed using 35ul of 1.18mM sample and a 1.7mm microcryoprobe. The structure was determined using triple resonance NMR spectroscopy. Automated backbone assignments were made using AutoAssign. Sidechain assignments were completed manually. Automated NOESY assignments were made using AutoStructure and structure solution was determined using AutoStructure and CYANA-2.1. The structure calculations were done including the C-terminal 6xHis tag LEHHHHHH. Resonance assignments were validated using AVS validation software. |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | chemical shift assignment | AutoAssign | 2.2.1 | Zimmerman, Moseley, Kulikowski and Montelione |
| 2 | structure solution | AutoAssign | 2.2.1 | Zimmerman, Moseley, Kulikowski and Montelione |
| 3 | chemical shift assignment | AutoStructure | 2.2.1 | Huang, Tejero, Powers and Montelione |
| 4 | structure solution | AutoStructure | 2.2.1 | Huang, Tejero, Powers and Montelione |
| 5 | geometry optimization | CYANA | 2.1 | Guntert, Mumenthaler and Wuthrich |
| 6 | structure solution | CYANA | 2.1 | Guntert, Mumenthaler and Wuthrich |
| 7 | refinement | CNS | 2.0.6 | Brunger, Adams, Clore, Gros, Nilges and Read |
| 8 | geometry optimization | CNS | 2.0.6 | Brunger, Adams, Clore, Gros, Nilges and Read |
