Calculations of free energy changes (ΔΔG) of hASIC1a substitutions. (A–F) Free energy changes (ΔΔG) calculations of hASIC1a substitutions of F21 (A), Y456 (B), E452 (C), W46 (D), H28 (E), and D455 (F) in the closed, open, and desensitized states compared with the WT hASIC1a in the corresponding conformations. A ΔΔG value >1 REU is considered to be destabilizing, while a ΔΔG value less than −1 REU is considered to be stabilizing. All values are presented as mean ± SD. 10 runs of free energy calculations were conducted in replicates for each mutant and the WT hASIC1; 10 free energy values of the WT and mutants were sorted from smallest to largest before free energy values of the WT were subtracted from those of each mutant (10 ΔΔG values obtained for each mutant). Each error bar shows the SD of 10 ΔΔG values. For each amino acid substitution and the WT hASIC1a, the total free energy score was calculated from contributions of the individual score terms in the RosettaMembrane energy function to the stability of the closed, open, and desensitized state structures, respectively. Values of calculations are shown in Data S2. Breakdowns of Rosetta score terms applied in this study are listed in the following categories: LJ (Lennard-Jones), LK and Elec nonbonded: fa_atr, fa_rep, fa_sol, fa_intra_rep, fa_intra_sol_xover4, lk_ball_wtd, fa_elec; hydrogen bonding: hbond_sr_bb, hbond_lr_bb, hbond_bb_sc, hbond_sc; disulfide: dslf_fa13; backbone and sidechain geometry: omega, fa_dun, p_aa_pp, yhh_planarity, ref, sugar_bb, rama_prepro, cart_bonded. Detailed individual Rosetta score terms can be found at https://new.rosettacommons.org/docs/latest/rosetta_basics/scoring/score-types.