Theoretically, the free energy cost to evolve from different thermodynamics states (i.e., fused bilayers, nucleation or expansion) must be independent of the direction of the collective variable. Therefore, the opening and closing paths of the fusion pore must be identical in the free energy profile. Any significant differences between them suggest hysteresis problems, inadequate sampling and/or poor convergence. To check for hysteresis-free sampling, initial configurations in both directions in the space of ξ e (forward and backward) were taken from a slow-growth path, as originally suggested by Pearlman and Kollman. Profiles in figure show no significant hysteresis.