2A63
Solution structure of a stably monomeric mutant of lambda Cro produced by substitutions in the ball-and-socket interface
SOLUTION NMR
NMR Experiment | ||||||||
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Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
1 | 3D_15N-separated_NOESY | 5 mM lambda Cro A33W/F58D/Y26Q U-15N, 50mM Na-phosphate, 90% H2O, 10% D2O, 0.01% sodium azide, 1 mM TSP | 90% H2O/10% D2O | no salt added | 5.3 | ambient | 293 | |
2 | HNHA | 5 mM lambda Cro A33W/F58D/Y26Q U-15N, 50mM Na-phosphate, 90% H2O, 10% D2O, 0.01% sodium azide, 1 mM TSP | 90% H2O/10% D2O | no salt added | 5.3 | ambient | 293 | |
3 | HNHB | 5 mM lambda Cro A33W/F58D/Y26Q U-15N, 50mM Na-phosphate, 90% H2O, 10% D2O, 0.01% sodium azide, 1 mM TSP | 90% H2O/10% D2O | no salt added | 5.3 | ambient | 293 | |
4 | HSQC (amide hydrogen exchange) | 5 mM lambda Cro A33W/F58D/Y26Q U-15N, 50mM Na-phosphate, 0.01% sodium azide, 1 mM TSP | 100% D2O | no salt added | 5.3 | ambient | 293 | |
5 | 3D_13C-separated_NOESY | 2.5 mM lambda Cro A33W/F58D/Y26Q U-13C, 50mM Na-phosphate, 90% H2O, 10% D2O, 0.01% sodium azide, 1 mM TSP | 90% H2O/10% D2O | no salt added | 5.3 | ambient | 293 | |
6 | 2D NOESY | 5 mM lambda Cro A33W/F58D/Y26Q unlabelled, 50mM Na-phosphate, 90% H2O, 10% D2O, 0.01% sodium azide, 1 mM TSP | 90% H2O/10% D2O | no salt added | 5.3 | ambient | 293 | |
7 | 2D NOESY | 5 mM lambda Cro A33W/F58D/Y26Q unlabelled, 50mM Na-phosphate, 90% H2O, 10% D2O, 0.01% sodium azide, 1 mM TSP | 90% H2O/10% D2O | no salt added | 6.1 | ambient | 293 | |
8 | 2D NOESY | 5 mM lambda Cro A33W/F58D/Y26Q unlabelled, 50mM Na-phosphate, 90% H2O, 10% D2O, 0.01% sodium azide, 1 mM TSP | 90% H2O/10% D2O | no salt added | 6.1 | ambient | 298 | |
9 | 2D NOESY | 5 mM lambda Cro A33W/F58D/Y26Q U-15N, 50mM Na-phosphate, 0.01% sodium azide, 1 mM TSP | 100% D2O | no salt added | 5.3 | ambient | 293 |
NMR Spectrometer Information | |||
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Spectrometer | Manufacturer | Model | Field Strength |
1 | Bruker | DRX | 600 |
NMR Refinement | ||
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Method | Details | Software |
simulated annealing | 40 structures were calculated using 838 noe-derived restraints, 14 hydrogen bond distance restraints, 50 phi angle restraints and 17 chi1 angle restraints. All 40 calculations converged to structures with no noe violations > 0.5 angstroms and no dihedral angle restraint violations >5 degrees. 14 of the 40 structures were discarded based on incompatibility of the rotamer of val 55 with a small J(HNHB) coupling constant, though no explicit restraint on the chi1 angle was included in the calculation. Of the 26 remaining structures, the 6 with the highest energy were discarded. In addition to having high energies, these 6 structures showed unusual conformations, particularly in turn regions, that were unreasonable and in strong disagreement with previously published structures of lambda Cro variants. The final ensemble contains 20 members. The ordered region of the protein extends from approximately residue 3 to residue 55. Pairwise RMSDs for the ordered region were 0.66 A (backbone atoms) and 1.29 A (all heavy atoms). None of the backbone angles in the ordered region of any ensemble member fell outside the most favorable and additionally allowed regions of a ramachandran plot. | XwinNMR |
NMR Ensemble Information | |
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Conformer Selection Criteria | all structures compatible with experimental restraints; structures chosen had the lowest energies and/or best agreement to J(NHB) data not used in explicit restraints |
Conformers Calculated Total Number | 40 |
Conformers Submitted Total Number | 20 |
Representative Model | 1 (closest to the average) |
Additional NMR Experimental Information | |
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Details | Proton chemical shifts submitted with this deposition were referenced to TSP at 0.00 ppm. However, we should add a cautionary note that this referencing led to an unusually high value for the water signal of 4.966 at 293 K, pH 5.3. We suspect that the TSP resonance is shifted below 0 ppm in our samples, possibly as much as -0.15 ppm. In our view, this is probably due to some transient interaction of the standard with the protein. |
Computation: NMR Software | ||||
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# | Classification | Version | Software Name | Author |
1 | collection | XwinNMR | 3.1 | Bruker |
2 | processing | NMRPipe | 1.8 | Frank Delaglio, Stephan Grzesiek, Ad Bax, Guang Zhu, Geerten Vuister, John Pfeifer |
3 | data analysis | NMRView | 4.1.3 | Bruce Johnston |
4 | structure solution | CNS | 1.1 | |
5 | refinement | CNS | 1.1 |