Fig 2. (a) Perturbed Helmholtz free-energy changes (useful work) of single K⁺ and Na⁺ ions pulled through the Na_vAb channel with relaxation times equal to 0.5 ns for each pulling step. The value of λ indicates the center of the harmonic pulling potential (see Fig 1). (b) Averaged z-coordinate of single K⁺ and Na⁺ ions. The z-coordinates more negative than –15 Å are on the extracellular side of the membrane, and z-coordinates greater than 10 Å are on the intracellular of the membrane.