TABLE 1.
Summary of simulations
| Label | tsim (ns) | Type | Ensemble | γ (ps−1) | Velocity (nm/ns) | Start |
|---|---|---|---|---|---|---|
| SimCa1 | 10.00 | EQ | NpT/NVE* | — | — | — |
| SimCa2 | 0.67 | PCV | NV | — | 5 × 2 | SimCa1 (5.0 ns) |
| SimCa2E | 1.07 | PCV | NV | — | 5 × 2 | SimCa2 |
| SimCa3 | 10.00 | REL | NVE | — | — | SimCa2 |
| SimCa4 | 4.04 | EQ | NpT | 5.0 | — | SimCa1 (1.1 ns) |
| SimCa5 | 1.70 | PCV | NpT | 5.0 | 5 × 2 | SimCa4 |
| SimCa6 | 2.15 | PCV | NpT | 0.1 | 5 × 2 | SimCa4 |
| SimCa7 | 0.50 | PCV | NV | — | 50 × 2 | SimCa1 (5.0 ns) |
| SimCa8 | 0.50 | PCV | NV | — | 25 × 2 | SimCa1 (5.0 ns) |
| SimCa9 | 14.96 | PCV | NV | — | 0.5 × 2 | SimCa1 (5.0 ns) |
| SimCa10 | 1.30 | PCV† | NV | — | 5 × 2 | SimCa1 (5.0 ns) |
| SimCa11 | 1.30 | PCV† | NV | — | 5 × 2 | SimCa1 (5.0 ns) |
| SimCa12 | 5.00 | PCL | NV | — | 10/13 nm/C | SimCa1 (5.0 ns) |
| SimCa13 | 5.00 | PCL | NV | — | 10/8 nm/C | SimCa1 (5.0 ns) |
| SimCa14 | 3.60 | PCL | NV | — | 10/15 nm/C | SimCa1 (5.0 ns) |
| SimCa15 | 2.90 | PCL | NV | — | 10/13 nm/N | SimCa1 (5.0 ns) |
| SimCa16 | 2.30 | PCL | NV | — | 10/14 nm/N | SimCa1 (5.0 ns) |
| SimCa17 | 3.10 | PCL | NV | — | 10/8 nm/N | SimCa1 (5.0 ns) |
| SimApo1 | 10.00 | EQ | NpT/NVE* | — | — | — |
| SimApo2 | 0.65 | PCV | NV | — | 5 × 2 | SimApo1 (5.0 ns) |
| SimApo2E | 1.05 | PCV | NV | — | 5 × 2 | SimApo2 |
| SimApo3 | 5.00 | REL | NVE | — | — | SimApo2 |
| SimApo4 | 9.34 | EQ | NpT | 5.0 | — | SimApo1 (1.1 ns) |
| SimApo5 | 14.61 | EQ | NpT | 0.1 | — | SimApo1 (1.1 ns) |
| SimApo6 | 2.00 | PCV | NpT | 5.0 | 5 × 2 | SimApo1 (5.0 ns) |
| SimApo7 | 2.10 | PCV | NpT | 0.1 | 5 × 2 | SimApo1 (5.0 ns) |
| SimApo8 | 12.10 | PCV | NV | — | 0.5 × 2 | SimApo1 (5.0 ns) |
| SimApo9 | 3.40 | PCL | NV | — | 10/8 nm/C | SimApo1 (5.0 ns) |
| SimK1 | 10.00 | EQ | NpT/NVE* | — | — | — |
| SimK2 | 5.00 | EQ | NpT/NVE* | — | — | — |
| SimNa1 | 10.00 | EQ | NpT/NVE* | — | — | — |
| SimNa2 | 1.77 | PCV | NV | — | 5 × 2 | SimNa1 (5.0 ns) |
Labels indicate the presence (Ca) or absence (Apo) of crystallographic Ca2+ ions in the system. Replacement of Ca2+ by Na+ or K+ is indicated by labels Na and K, respectively. EQ denotes equilibrium simulations, PCV denotes constant velocity SMD simulations, and REL denotes free dynamics simulations in the corresponding ensemble. PCL denotes constant velocity SMD simulations in which one end of the protein is held fixed while the other end of the protein (N- or C-terminus) is pulled until a predefined elongation has being achieved. Then, the steering atom is held in space and the protein is allowed to relax in a so-called length-clamp steering protocol (see Methods). Initial coordinates and velocities were obtained from the last frame of the simulations mentioned in the Start column. All SMD simulations were performed by attaching steering springs to Cα atoms of residues 1 and 540, unless otherwise stated.
These simulations consisted of 1000 steps of minimization, 100 ps of dynamics with the backbone of the protein restrained (k = 1 Kcal/mol/Å2), and the remaining time as free dynamics in the NpT (1 ns with γ = 5 ps−1) and NVE ensembles.
These SMD simulations were performed by pulling repeats one and three (SimCa10) or three and five (SimCa11) in opposite directions (see Methods).