2HEP
Solution NMR structure of the UPF0291 protein ynzC from Bacillus subtilis. Northeast Structural Genomics target SR384.
SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | 3D_15N-separated_NOESY | 1.12mM U-13C,15N SR384, 20mM MES, 100mM NaCl, 5 mM CaCl2, 10mM DTT, 0.02% NaN3, pH 6.5 | 5% D2O/95% H2O | 100 mM NaCl | 6.5 | ambient | 293 | |
| 2 | 3D_13C-separated_NOESY | 1.12mM U-13C,15N SR384, 20mM MES, 100mM NaCl, 5 mM CaCl2, 10mM DTT, 0.02% NaN3, pH 6.5 | 5% D2O/95% H2O | 100 mM NaCl | 6.5 | ambient | 293 | |
| 3 | HNHA | 1.12mM U-13C,15N SR384, 20mM MES, 100mM NaCl, 5 mM CaCl2, 10mM DTT, 0.02% NaN3, pH 6.5 | 5% D2O/95% H2O | 100 mM NaCl | 6.5 | ambient | 293 | |
| 4 | 3D GFT-CBCACAcoNHN, GFT-HNNCACBCA, GFT-HABCABcoNHN | 1.12mM U-13C,15N SR384, 20mM MES, 100mM NaCl, 5 mM CaCl2, 10mM DTT, 0.02% NaN3, pH 6.5 | 5% D2O/95% H2O | 100 mM NaCl | 6.5 | ambient | 293 | |
| 5 | 3D HCCH-TOCSY, CCcoNH TOCSY, HCCH-COSY | 1.12mM U-13C,15N SR384, 20mM MES, 100mM NaCl, 5 mM CaCl2, 10mM DTT, 0.02% NaN3, pH 6.5 | 5% D2O/95% H2O | 100 mM NaCl | 6.5 | ambient | 293 | |
| 6 | high resolution 2D CH-HQSC (for stereospecific assignment of Val/Leu methyls) | 1.12mM 5%-13C,U-15N SR384, 20mM MES, 100mM NaCl, 5 mM CaCl2, 10mM DTT, 0.02% NaN3, pH 6.5 | 5% D2O/95% H2O | 100 mM NaCl | 6.5 | ambient | 293 | |
| 7 | 3D HNCO, HNcaCO | 1.12mM U-13C,15N SR384, 20mM MES, 100mM NaCl, 5 mM CaCl2, 10mM DTT, 0.02% NaN3, pH 6.5 | 5% D2O/95% H2O | 100 mM NaCl | 6.5 | ambient | 293 | |
| 8 | 2D 15N1H-heteronuclear NOE | 1.12mM U-13C,15N SR384, 20mM MES, 100mM NaCl, 5 mM CaCl2, 10mM DTT, 0.02% NaN3, pH 6.5 | 5% D2O/95% H2O | 100 mM NaCl | 6.5 | ambient | 293 | |
| NMR Spectrometer Information | |||
|---|---|---|---|
| Spectrometer | Manufacturer | Model | Field Strength |
| 1 | Varian | INOVA | 600 |
| 2 | Bruker | AVANCE | 600 |
| NMR Refinement | ||
|---|---|---|
| Method | Details | Software |
| simulated annealing | The structures are based on a total of 403 conformationally restricting NOE-derived distance constraints, 67 dihedral angle constraints, 42 hydrogen bond constraints, and 29 J(HN-Halpha) coupling constants (12.5 constraints per residue; 1.1 long range constraints per residue; computed for residues 1 to 42 by PSVS 1.2). Structure determination was performed iteratively using AutoStructure (XPLOR-NIH). A final XPLOR calculation using the final constraints derived from AutoStructure was performed to yield the final structures. The unstructured C-terminal half of the molecule was included in all structural calculations but has been omitted from this deposition. Heteronuclear 15N,1H NOE experiments corroborate the presence of significant disorder in the C-terminal half of the protein (residues 43 to C-terminus). | VNMR |
| NMR Ensemble Information | |
|---|---|
| Conformer Selection Criteria | structures with lowest conformational energy |
| Conformers Calculated Total Number | 100 |
| Conformers Submitted Total Number | 20 |
| Representative Model | 1 (lowest energy) |
| Additional NMR Experimental Information | |
|---|---|
| Details | The structure was determined using triple resonance NMR spectroscopy. AUTOASSIGN was used to obtain automatic backbone assignments from GFT peak lists. Side chain assignments were completed manually. Automatic NOESY assignment, as well as distance and hydrogen bond constraints were determined using AutoStructure. Dihedral angle constraints were determined using HYPER. Completeness of NMR assignments (excluding C-terminal LEHHHHHH tag): Backbone: 91.9%; Side chain: 78.3%; Aromatics, 100%: Stereospecific methyl, 87.5%. Final structure quality factors (for residues 1 to 42 only; PSVS 1.2), where ordered residues [S(PHI)+ S(PSI) > 1.8] comprise 5-20 and 23-40: (A) RMSD (ordered residues): BB, 0.9; heavy atom, 1.5. (B) Ramachandran statistics for ordered residues: Most favored, 96.4%, Additionally allowed, 3.6%, generously allowed, 0.0%, disallowed, 0.0%. (C) Procheck scores for ordered residues (Raw/Z-): PHI-PSI, 0.11/0.75; All, -0.12/-0.71. (D) Molprobity clash score (Raw/Z-): 14.69/-1.00. (E) RPF scores for goodness of fit to NOESY data (all residues): Recall, 0.952, Precision, 0.839, F-measure, 0.892, DP-score, 0.628 |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | collection | VNMR | 6.1C | Varian |
| 2 | collection | XwinNMR | 3.5pl6 | Bruker |
| 3 | data analysis | AutoAssign | 2.2.1 | Zimmerman, Moseley, Montelione |
| 4 | data analysis | Sparky | 3.110 | Goddard & Kneller |
| 5 | refinement | AutoStructure | 2.1.1 | Huang & Montelione |
| 6 | refinement | XPLOR-NIH | 2.11.2 | Clore et al |
| 7 | data analysis | AGNuS | 2.0 | Moseley & Montelione |
| 8 | data analysis | PdbStat | 4.01 | Tejero & Montelione |
| 9 | data analysis | PSVS | 1.2 | Bhattacharya & Montelione |
