THREE-DIMENSIONAL STRUCTURE OF THE PENTAMERIC HELICAL BUNDLE OF THE ACETYLCHOLINE RECEPTOR M2 TRANSMEMBRANE SEGMENT
SOLID-STATE NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | HSQC; HNCA; HNCOCA; 3D 15N- and 13C-edited NOESY; 3D 15N- and 13C-edited TOCSY; 3D HNHA | 1mM M2 U-15N; 350mM DPC; pH5.5; | 90% H2O/10% D2O | 5.5 | ambient | 303 | ||
| 2 | 1D CPMOIST; 2D 1H/1H-15N PISEMA; 2D 1H/15N HETCOR; | 20mg M2 U-15N; 40mg DMPC; | DMPC bilayers in H2O; | 5.5 | ambient | 298 | ||
| NMR Spectrometer Information | |||
|---|---|---|---|
| Spectrometer | Manufacturer | Model | Field Strength |
| 1 | Bruker | DMX | 750 |
| 2 | Bruker | DMX | 600 |
| 3 | Chemagnetics | CMX | 400 |
| NMR Refinement | ||
|---|---|---|
| Method | Details | Software |
| X-PLOR 3.1, distance geometry, simulated annealing, molecular dynamics, FISI, FINGERPRINT, HOLE | The backbone coordinates obtained from solution NMR were superimposed on the coordinates obtained from solid-state NMR to fix the helix orientation and rotation in the membrane. The pentameric array was then optimized using molecular dynamics. Pore contours were calculated with the program HOLE. | X-PLOR |
| NMR Ensemble Information | |
|---|---|
| Conformer Selection Criteria | structures with favorable non-bond energy |
| Conformers Calculated Total Number | 30 |
| Conformers Submitted Total Number | 1 |
| Representative Model | (minimized average structure) |
| Additional NMR Experimental Information | |
|---|---|
| Details | The orientation of each monomer in the pentameric bundle was obtained from the combination of the solution NMR (PDB file 1a11) and solid-state NMR (PDB file 1cek) structures |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | refinement | X-PLOR | 3.01 | Brunger |
| 2 | structure solution | FISI | 1.01 | Marassi |
| 3 | structure solution | HOLE | Smart, Goodfellow, Wallace | |
| 4 | data analysis | Felix | 95 | |
