The summary of the protocol of the EM-fitting and umbrella sampling simulations.

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. Rf was defined as the average position of R, <R>W = N in the N-th window.