Table 1. The summary of the protocol of the EM-fitting and umbrella sampling simulations.
| Simulation | Target EMD | Number of windows | Simulation time for each window [ns] | Simulation time [ns] | Number of windows used for free-energy calculation | Correspondences with W (the window number in Fig. S2), and the definition of Rf | Inner product between Rf and Rf in the “reverse translocation” |
| 1. Initial equilibration | 5.5 | ||||||
| 2. “classical tRNA” in Fig. S2(a) | 1365 | 42 | 0.5a+1.0b+2.0c = 3.5 | 147.0 | 42 | W = 1–42, R f = <R>W = 42 (R1(R f) = 30.8 Å) | 0.910 |
| 3. “classical tRNA” in Fig. S2(b) | 1363 | 18 | 3.5 | 63.0 | 14A+18 = 32 | W = 15–32, R f = <R>W = 32 (<R>W = 14 = 14.0 Å) | 0.904 |
| 4. “classical tRNA” in Fig. S2(c) | 1363 | 12 | 3.5 | 42.0 | 20B+12C = 32 | W = 21–32, R f = <R>W = 32 (<R>W = 20 = 18.3 Å) | 0.938 |
| 5. “classical tRNA” in Fig. S2(d) | 1363 | 7 | 3.5 | 24.5 | 25D+7 = 32 | W = 26–32, R f = <R>W = 32 (<R>W = 25 = 21.9 Å) | 0.906 |
| 6. “classical tRNA” in Fig. S2(e) (or Fig. S4(a)) | 1363 | 25 | 3.5 | 87.5 | 20B+12C+25 = 57 | W = 33–57, R f = <R>W = 46 (R1(R f) = 36.1 Å) | 0.957 |
| 7. Modeling “semi-hybrid tRNA” | 10.0 | ||||||
| 8. “semi-hybrid tRNA” in Fig. S4(b) | 1365 and 1363 | 6 and 23 | 3.5 | 101.5 | 29 | (Figure not shown) | |
| 9. Modeling “hybrid tRNA” | 10.0 | ||||||
| 10. “hybrid tRNA” in Fig. S4(c) | 1363 | 16 | 3.5 | 56.0 | 16 | (Figure not shown) | |
| 11. “r-translocation” in Fig. 2 (or Fig. S2(f)) | 1365 and 1363 | 10 and 38 | 0.5a+2.0b+2.0c = 4.5 | 216.0 | 14A+10+38 = 62 | W = 15–24, 25–62, R f = <R>W = 52 (<R>W = 24 = 18.5 Å, R1(R f) = 36.9 Å) | 1.000 |
| 12. “r-translocation” (second run) in Fig. 2 (or Fig. S2(g)) | 1365 and 1363 | 10 and 33 | 3.5 | 150.5 | 14A+10+33 = 57 | W = 15–24, 25–57, R f = <R>W = 50 (<R>W = 24 = 19.8 Å, R1(R f) = 36.5 Å) | 0.969 |
| 13. effect of different ion force-fields in Fig. S2(h) | 33 | 3.5 | 115.5 | 14A+10E+33 = 57 | |||
| 14. effect of different umbrella variables in Fig. S7 | 32.0 | ||||||
| Total | 1061.0 |
a, b and c in “Simulation time for each window” are the simulation times for the EM-fitting, equilibration and umbrella sampling simulations, respectively. A, B, C and D in “Number of windows used for free-energy calculation”, which were out of 42 windows obtained at “2. “classical tRNA” in Fig. S2 (a)”, were reused for each free-energy calculation. E in “Number of windows used for free-energy calculation”, which was obtained at “12. “r-translocation” (second run) in Fig. 2 (or Fig. S2(g))”, were reused for the free-energy calculation using a corrected ion force-field. R f was defined as the average position of R, <R>W = N in the N-th window.