domains is in between the other two cases. We hypothesize that the ratio between the amounts of water molecules in the channel and lipids surrounding it, is optimized for 2 Syt1-C2B domains to release free energy and to lower the nucleation expansion barrier ΔG ne (see figure ). Also, we have observed that 2 Syt1-C2B domains increase the amount of PIP 2 :PIP 2 interactions with respect to systems with 1 Syt1-C2B domain and membranes-only (for details, see supporting information). This increased propensity to form PIP 2 clusters has been observed before and is in agreement with other studies pointing to PIP 2 clusters as molecular beacons during vesicle recruitment. lipid molecules Therefore, the presence of one or two Syt1-C2B domains ensures that a nascent fusion pore at ξ e ∼ 0 will (most probably) evolve towards meta-stabilization at ξ e ∼ 0.4. Once there, the reduced -expansion barrier ΔG ne for two C2B domains facilitates its exit from the metastable state. Together with the reduced cost for expansion (ξ e > 0.4), the system containing a pair of C2B domains naturally evolves in the most probable direction of fusion pore expansion, therefore, significantly reducing the probability of pore resealing and kissand-run events.