As shown before, the energy barrier for the first stalk is ΔG s ∼150 kJ/mol for an identical pair of POPC:POPS:PIP 2 bilayers (87.5:10:2.5) in the Martini 3 space, using SAP2 model for PIP 2 lipids. The energy barrier ΔG n ∼350 kJ/mol was also calculated before, for a similar pair of POPC:POPS:PIP 2 bilayers (87.5:10:2.5) in the Martini 2 space using POP2 model for PIP 2 lipids. Stabilization and expansion of the nucleated fusion pore were calculated in this work using Martini 3 for a pair of POPC:POPS:PIP 2 bilayers (87.5:10:2.5) in the Martini 3 space, using also SAP2 model Figure : The life of the fusion pore. Schematic PMF for stalk formation, fusion pore meta-stabilization and expansion. Solid lines are effective PMF calculations from umbrella sampling simulations, while regions not completely described yet are indicated with dashed lines. In all cases, data corresponds to membranes-only POPC:POPS:PIP 2 systems. pores easily fall in the expansion region (to the right in figure , colored in green) or reseal returning to the stalk (to the left, in figure 4, colored in lilac). Previously, we have studied the nucleation of the fusion pore with negligible hysteresis (represented by the solid line in the pink zone of figure ). However, a slight widening of the water channel produce unstable configurations that cannot be sustained over long simulation times (dashed line in the pink zone in figure ). Such behavior prevents adequate sampling in the nucleation region and discourages all attempts to calculate the free energy profile, further in the space of ξ n . Therefore, once we generated a nucleated fusion pore in the space of ξ n we have let it evolve freely in the space of ξ e where it could be later controlled, either to be shrunk or enlarged. The general shape of the schematic PMF shown in figure is in good agreement with other proposed energy landscapes. The Synaptotagmin-1 C2B domains reduce the free energy for fusion pore expansion