Once there, the metastable fusion pore may continue to expand or it may reseal, still allowing the vesicle to partially deliver its contents (kiss-and-run). If resealing, the fusion pore may flicker, opening and shutting multiple times before expanding again or before definitively closing. Here, for POPC:POPS:PIP 2 bilayers in the Martini 3 coarse-grained space, we report a nucleation-expansion barrier of ΔG ne ∼100 kJ/mol (∼40k B T ), see figs. Lower values of ΔG ne indicate that the metastable fusion pore at ξ e ∼ 0.4 may more easily reconfigure back to a nucleated (and hence unstable) pore at ξ e ∼ 0. Remarkably, Syt1-C2B domains have significant effects on this energy barrier, independently of the effects on expansion (ξ e > 0.4) described in the previous section. Red line in figure shows that one C2B domain marginally increases the nucleation-expansion energy barrier (ΔG ne ), see region 0 < ξ e < 0.4. However, the opposite effect is observed for a pair of C2B domains surrounding the fusion pore. Blue line shows that ΔG ne ∼ 60kJ/mol, a reduction to almost one-half with respect to membranes-only system. Such collaborative behavior between a pair of C2B domains has already been reported for the fusion stalk. However, C2B domains not only modify the nucleation-expansion barrier ΔG ne but also the unexplored nucleation region (figure , dashed line in the pink zone). We have run 1,000 independent 5ns unbiased simulations from the same initial configuration: a nascent fusion pore at ξ e ∼ 0. We have conducted this procedure for the three systems: membranesonly system (black), with one Syt1-C2B (red) and with two Syt1-C2B (blue). Figure concentrates 15 µs of data from molecular dynamics for the three systems. Statistically, it can be observed that the membranes-only system shows the lowest values of ξ e (even ξ e < 0), indicating the existence of trajectories that evolve in the direction of pore closure and membrane recovery (black). On the contrary, both systems including one (red) or two (blue) Syt1-C2B domains show only positive values of ξ e with all trajectories evolving toward meta-stabilization at ξ e ∼ 0.4 (see panels 6a and 6b).