Relative and intrinsic binding free energies in KcsA from MD simulations and experiments (kcal mol⁻¹).

Protein	Conformation	AMOEBA		ITCd
		K+	Na+ → K+	K+	Na+ → K+
KcsA-WT	Collapsed	−4.9 ± 0.7	−1.6 ± 0.3b	−4.7	−2.1
KcsA-WT	Conductive	−6.8 ± 0.6a	−2.0 ± 0.2c	−5.7	−2.9
		−1.3 ± 0.6
KcsA-G77A	Conductive	−6.0 ± 0.5	−3.3 ± 0.4b

^aThe two values from top to bottom are free energy changes from 2 K⁺ to 3 K⁺ and 3 K⁺ to 4 K⁺ in the SF, respectively. 3 K⁺ free energy is calculated by sum of partition functions of all four configurations. 2 K⁺ free energy is from the water–K⁺–water–K⁺ configuration.

^bFree energy change from 2 Na⁺ to 1 Na⁺/1 K⁺ in the SF.

^cFree energy change from 1 Na⁺/3 K⁺ to 4 K⁺ in the SF.

^dExperimental binding free energy was calculated from ΔG = −RT ln[K_d/(mol L⁻¹)], with K_d(K⁺) = 67 and 350 μM, K_d(Na⁺) = 9 and 0.48 mM in wildtype (conductive) and M96V mutant (collapsed) KcsA, respectively.⁴⁷