Table 1.

Primary free energy data

Process	TI forward (kJ/mol)	TI reverse (kJ/mol)	HRE (kJ/mol)
ΔG Mut	19.7 ± 2.4	−0.7 ± 3.2	8.8 ± 2.9
ΔG Mut_R	23.1 ± 3.3	−20.6 ± 3.3	24.4 ± 2.3
ΔG Mut_F	29.3 ± 1.6	−10.0 ± 2.6	–
ΔG Mut_S	18.1 ± 2.2	−17.0 ± 2.5	21.2 ± 2.2
ΔG IRF	−9.1 ± 0.3	8.9 ± 0.5	−8.9 ± 0.3
ΔG IFS	8.6 ± 0.5	−8.8 ± 0.5	8.6 ± 0.3
ΔG IRF_wt	−1.1 ± 0.7	3.8 ± 0.8	−2.1 ± 0.6
ΔG IFS_wt	14.4 ± 1.3	−17.8 ± 1.1	9.2 ± 0.9
ΔG IRF_M	−4.6 ± 0.7	4.7 ± 0.8	−6.5 ± 0.7
ΔG IFS_M	7.6 ± 0.9	−4.4 ± 0.8	8.0 ± 0.7

Free energy differences for all processes of the thermodynamic scheme in Fig. 4 for forward (ΔG ^for) and reverse (ΔG ^rev) TI and Hamiltonian replica exchange (ΔG ^hre) schemes, and their statistical uncertainty after 1 ns per λ point simulation time