Solution NMR structure of the ydfO protein from Escherichia coli. Northeast Structural Genomics target ER251.
SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | 3D_13C-separated_NOESY | 0.69mM U-13C,15N ER251, 0.02% NaN3, 10mM DTT, 5mM CaCl2, 100mM NaCl, 20mM Ammonium acetate, 5% D2O | 5% D2O, 95% H2O | 0.1 M NaCl | 4.5 | atmospheric atm | 293 | |
| 2 | 3D_15N-separated_NOESY | 0.69mM U-13C,15N ER251, 0.02% NaN3, 10mM DTT, 5mM CaCl2, 100mM NaCl, 20mM Ammonium acetate, 5% D2O | 5% D2O, 95% H2O | 0.1 M NaCl | 4.5 | atmospheric atm | 293 | |
| 3 | HcCH-TOCSY, HcCH-COSY,CccoNH-TOCSY | 0.69mM U-13C,15N ER251, 0.02% NaN3, 10mM DTT, 5mM CaCl2, 100mM NaCl, 20mM Ammonium acetate, 5% D2O | 5% D2O, 95% H2O | 0.1 M NaCl | 4.5 | atmospheric atm | 293 | |
| 4 | 3D-HNCACB, HNcoCACB, HBHAcoNH, HNCO | 0.69mM U-13C,15N ER251, 0.02% NaN3, 10mM DTT, 5mM CaCl2, 100mM NaCl, 20mM Ammonium acetate, 5% D2O | 5% D2O, 95% H2O | 0.1 M NaCl | 4.5 | atmospheric atm | 293 | |
| 5 | C13_HSQC_noct Stereospecific VL Me assign. | 0.74mM 5%-13C,U-15N ER251 0.02% NaN3, 10mM DTT, 5mM CaCl2, 100mM NaCl, 20mM Ammonium acetate, 5% D2O | 5% D2O, 95% H2O | 0.1 M NaCl | 4.5 | atmospheric atm | 293 | |
| 6 | Het-NOE, T1/T1rho | 0.74mM 5%-13C,U-15N ER251 0.02% NaN3, 10mM DTT, 5mM CaCl2, 100mM NaCl, 20mM Ammonium acetate, 5% D2O | 5% D2O, 95% H2O | 0.1 M NaCl | 4.5 | atmospheric atm | 293 | |
| 7 | 3D_13C-separated_NOESY | 0.69mM U-13C,15N ER251, 0.02% NaN3, 10mM DTT, 5mM CaCl2, 100mM NaCl, 20mM Ammonium acetate, 5% D2O | 99.9% D2O | 0.1 M NaCl | 4.5 | atmospheric atm | 293 | |
| NMR Spectrometer Information | |||
|---|---|---|---|
| Spectrometer | Manufacturer | Model | Field Strength |
| 1 | Bruker | AVANCE | 800 |
| 2 | Bruker | AVANCE | 600 |
| 3 | Varian | INOVA | 750 |
| 4 | Varian | INOVA | 600 |
| NMR Refinement | ||
|---|---|---|
| Method | Details | Software |
| Noesy assignments made with iterative method using CS, 3J, Hyper (dihedral), and Dyana for simulated annealing MD. Converged structures are further refined using NIH-xplor followed by CNS in explicit water shell (Nielges) . Full lenght sequence was carried through the refinement protocol, the disordered regions and hexHIS tag are not reported. Structure based on 1885 constraints, 788 long range, 142 dihedral constraints and 114 H-bond constraints. | VNMR | |
| 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 | Structure determined by triple resonance NMR spectroscopy. Monomer under NMR conditions. TC = 9.1 +/-0.5 ns (1D T1/T1rho +/- FIT STD). Coordinates reported from residue 7 to 133 based on order parameter. AutoAssign used for backbone assignment, manually completed sidechain. 13C and 15N NOESY were assigned with AutoStructure. Dihedral angle restraints determined by HYPER . Assignment stats (excluding C-term tag): backbone 96.7%, sidechain 83.7%, aromatic (sc) 79.8%, VL methyl stereospecific 100%, unambiguos sidechain NH2 88.9%. Structure quality factor PSVS 1.3: ordered residues ranges alpha helix (8-22, 26-35, 76-88, 93-102), b-strand (39-43, 48-52, 58-62, 107-111, 116-120, 126-131) [S(phi)+S(psi)]>1.8. RMSD 0.5 bb, 1.1 all heavy atoms. Rama: 87.4% most fav, 12.5% addtl. all., 0.0 gen. all.,0.0% disall. Procheck (psi-phi): -0.17/-0.35 (raw/Z), Procheck (all): -0.15/-0.89 (raw/Z), MolProbity Clash: 24.43/-2.67 (raw/Z) . RPF scores all assigned residues (fit of noesy peaklists to structure): Recall: 0.965, Precision: 0.919, F-measure: 0.942, DP-score: 0.817. L139F cloning mutation of E. coli ydfO gene present. |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | collection | VNMR | 6.1C | Varian Inc. |
| 2 | collection | XwinNMR | 3.5 | Bruker Biospin |
| 3 | structure solution | DYANA | 1.2 | Gunthert |
| 4 | refinement | X-PLOR | 2.11.2 | Clore |
| 5 | processing | NMRPipe | 2005 | Delaglio |
| 6 | data analysis | Sparky | 3.11 | Goddard & Kneller |
| 7 | data analysis | AutoAssign | 2.2.1 | Zimmerman, Moseley, Montelione |
| 8 | structure solution | AutoStructure | 2.1.1 | Huang, Montelione |
| 9 | structure solution | HYPER | 2.1 | Tejero, Montelione |
| 10 | data analysis | PdbStat | 4.1 | Tejero, Montelione |
| 11 | refinement | PSVS | 1.3 | Bhattacharya, Montelione |
| 12 | refinement | CNS | 1.1 | Brunger |
| 13 | refinement | Procheck NMR | 3.51 | Laskowski, MacArthur |
| 14 | refinement | MolProbity | 3.01 | Lovell, Richardson et. al. |
