SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | HNCA | 1.2mM Stefin A U-15N; 50mM phosphate buffer pH 5.5, 100mM KCl, 5mM Azide; 90% H2O, 10% D2O | 90% H2O/10% D2O | 5.5 | ambient | 308 | ||
| 2 | HNCO | 1.2mM Stefin A U-15N; 50mM phosphate buffer pH 5.5, 100mM KCl, 5mM Azide; 90% H2O, 10% D2O | 90% H2O/10% D2O | 5.5 | ambient | 308 | ||
| 3 | HN(CA)CB | 1.2mM Stefin A U-15N; 50mM phosphate buffer pH 5.5, 100mM KCl, 5mM Azide; 90% H2O, 10% D2O | 90% H2O/10% D2O | 5.5 | ambient | 308 | ||
| 4 | TOCSY-HSQC | 1.2mM Stefin A U-15N; 50mM phosphate buffer pH 5.5, 100mM KCl, 5mM Azide; 90% H2O, 10% D2O | 90% H2O/10% D2O | 5.5 | ambient | 308 | ||
| 5 | NOESY-HSQC | 1.2mM Stefin A U-15N; 50mM phosphate buffer pH 5.5, 100mM KCl, 5mM Azide; 90% H2O, 10% D2O | 90% H2O/10% D2O | 5.5 | ambient | 308 | ||
| 6 | 15N-isotope-filtered NOESY | 1mM Stefin A U-15N,13C,2H; 50mM phosphate buffer pH 5.5, 100mM KCl, 5mM Azide; 90% H2O, 10% D2O | 90% H2O/10% D2O | 5.5 | ambient | 308 | ||
| NMR Spectrometer Information | |||
|---|---|---|---|
| Spectrometer | Manufacturer | Model | Field Strength |
| 1 | Bruker | DRX | 600 |
| 2 | Bruker | DRX | 800 |
| NMR Refinement | ||
|---|---|---|
| Method | Details | Software |
| simulated annealing | Distance restraints (NOEs and hydrogen bonds) were taken from the data used for the stefin A monomer structure (Martin et al., 1995), except those involving residues V48-G50 and N77-L80 where chemical shift perturbation was observed following resonance assignment. The NOEs were specified as intermolecular and intramolecular according to domain-swapped topology inferred from isotope filtering experiments. An additional hydrogen bond restraint was included between 48 and 50 based on evidence from protection experiments on the dimer (Jerala and Zerovnik, 1999). Dihedral restraints (phi and psi) were determined using the 1H-alpha, 15N, 13C-alpha, 13C-beta and 13C' chemical shifts and the program TALOS (Cornilescu et al., 1999). Where TALOS gave a `poor' match, the experimental phi dihedral angle was taken from the data used for the stefin A monomer structure calculation. | XwinNMR |
| NMR Ensemble Information | |
|---|---|
| Conformer Selection Criteria | |
| Conformers Calculated Total Number | |
| Conformers Submitted Total Number | 1 |
| Representative Model | 1 (minimized average structure) |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | collection | XwinNMR | 2.6 | Bruker |
| 2 | data analysis | Felix | 2000 | MSI |
| 3 | refinement | X-PLOR | 3.1 | BRUNGER |
