Comparison of nonadditivities evaluated with GIA and FMC from randomly sampled mutant cycles. (A–E) Several mutant cycles were generated via random sampling strategy. For each cycle, ΔΔG_FMC (A and D) and ΔΔG_GIA (B and E) were calculated and compared against ΔΔG_true. In A and B, each of the four constructs constituting the cycle follow a ZHA activation scheme, whereas in D and E, each of the four constructs constituting the cycle follow an MWC activation scheme (C), where J represents the intrinsic voltage-dependent activation constant of each voltage sensor, L is the intrinsic activation constant of the pore, and D is the allosteric interaction factor.