The presence of a single Syt1-C2B domain in the cytosolic space while membranes fuse and the fusion pore nucleates has shown negligible effects on the free energy profile. However, the simultaneous action of a pair of Syt1-C2B domains (or the tandem Syt1-C2AB) significantly reduced the free energy cost to form the fusion stalk. Here, we complete the scene by describing the C2B domain effects beyond nucleation and during the expansion of the fusion pore. Figure shows free energy profiles in the ξ e space starting from a nucleated pore (ξ e ∼ 0) to an expanded one (ξ e ∼ 0.75) for three cases: (i) membranes-only (black line), (ii) with a single Syt1-C2B domain in the cytosolic space (red line) and (iii) with a pair of Syt1-C2B domains also in the cytosolic space (blue line).