Solution structure of MerB, the Organomercurial Lyase involved in the bacterial mercury resistance system
SOLUTION NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | 3D 15N-edited NOESY-HSQC | 1.0-1.5mM MerB [U-15N], 10mM sodium phosphate buffer, 10mM sodium chloride, 7.5mM DTT, 1mM EDTA, 90%H2O, 10%D2O | 90% H2O/10% D2O | 10 mM sodium phosphate buffer and 10mM sodium chloride | 7.5 | ambient | 300 | |
| 2 | HNHA | 1.0-1.5mM MerB [U-15N], 10mM sodium phosphate buffer, 10mM sodium chloride, 7.5mM DTT, 1mM EDTA, 90%H2O, 10%D2O | 90% H2O/10% D2O | 10 mM sodium phosphate buffer and 10mM sodium chloride | 7.5 | ambient | 300 | |
| 3 | 3D 13C-edited HMQC-NOESY | 1.0-1.5mM MerB [U-13C and U-15N], 10mM sodium phosphate buffer, 10mM sodium chloride, 7.5mM DTT, 1mM EDTA | 99.9% D2O | 10 mM sodium phosphate buffer and 10mM sodium chloride | 7.5 | ambient | 300 | |
| 4 | 4D 13C/13C-edited HMQC-NOESY-HMQC | 1.0-1.5mM MerB [U-13C and U-15N], 10mM sodium phosphate buffer, 10mM sodium chloride, 7.5mM DTT, 1mM EDTA | 99.9% D2O | 10 mM sodium phosphate buffer and 10mM sodium chloride | 7.5 | ambient | 300 | |
| 5 | 2D IPAP-[1H-15N] HSQC | 1.3mM MerB [U-13C and U-15N], 10mM sodium phosphate buffer, 10mM sodium chloride, 7.5mM DTT, 1mM EDTA, 11 mg/ml Pf1 phages, 90%H2O, 10%D2O | 90% H2O/10% D2O | 10 mM sodium phosphate buffer and 10mM sodium chloride | 7.5 | ambient | 300 | |
| 6 | 3D TROSY-based HNCO for measuraments of one-bond dipolar couplings | 1.3mM MerB [U-13C and U-15N], 10mM sodium phosphate buffer, 10mM sodium chloride, 7.5mM DTT, 1mM EDTA, 11 mg/ml Pf1 phages, 90%H2O, 10%D2O | 90% H2O/10% D2O | 10 mM sodium phosphate buffer and 10mM sodium chloride | 7.5 | ambient | 300 | |
| NMR Spectrometer Information | |||
|---|---|---|---|
| Spectrometer | Manufacturer | Model | Field Strength |
| 1 | Varian | INOVA | 600 |
| 2 | Varian | INOVA | 800 |
| NMR Refinement | ||
|---|---|---|
| Method | Details | Software |
| Simulated annealing, with a combination of torsion angle dynamics and cartesian dynamics | The three-dimensional structures of MerB were determined using a set of 1493 NOE-derived distance restraints, 203 backbone dihedral angle (phi and psi) restraints, 5 chi angle restraints, 36 hydrogen-bond restraints and 298 one-bond residual dipolar coupling restraints. | VNMR |
| NMR Ensemble Information | |
|---|---|
| Conformer Selection Criteria | The submitted models are the 20 structures with no upper bound violation greater that 0.3 armstrongs and no dihedral angle restraint violation greater than 2 degrees and with the lowest energies. |
| Conformers Calculated Total Number | 55 |
| Conformers Submitted Total Number | 20 |
| Representative Model | 1 (among the structures with the lowest rmsd to the minimized averge structure, the model with the lowest energy was selected) |
| Additional NMR Experimental Information | |
|---|---|
| Details | The structure was determined using triple-resonance NMR spectroscopy. |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | collection | VNMR | Varian | |
| 2 | processing | NMRPipe | Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. and Bax, A. | |
| 3 | data analysis | PIPP | Garrett, D.S., Powers, R., Gronenborn, A.M., Clore, G.M. | |
| 4 | data analysis | NMRView | 5.0.4 | Johnson, B.A. and Blevins, R.A. |
| 5 | structure solution | CNS | 1.0 | Brunger, A.T., Adams, P.D., Clore, G.M., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. and Warren, G.L. |
| 6 | refinement | CNS | 1.0 | Brunger, A.T., Adams, P.D., Clore, G.M., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. and Warren, G.L. |
