To focus on the Syt1 component of the fusion clamp, all fusion analysis was carried out using vesicles containing low copy VAMP2 (~13 copies) with normal number (~25 copies) of Syt1 molecules (wild-type or targeted mutations) in the absence of Cpx. (A) Survival analysis shows that disrupting the Syt1-SNARE primary interface (Syt1Q, green curve) or destabilizing Syt1 oligomerization (Syt1F349A, purple curve) abrogates the Syt1 clamp, whilst the Syt1-Cpx-SNARE tripartite interface (Syt1LLQQ, yellow curve) and the Ca2+-binding motif on the Syt1 C2B domain (Syt1DA, red curve) are not involved in establishing the fusion clamp. (B) Addition of Ca2+ triggered rapid fusion of the majority (>90%) of Syt1LLQQ and the remainder (~15%) of the docked Syt1F349A vesicles, very similar to the behavior of the Syt1WT vesicles. Predictably, blocking Ca2+-binding to C2B domain rendered the vesicle Ca2+-insensitive, with the majority of Syt1DA remaining un-fused. We did not have sufficient number of docked Syt1Q vesicles to do a quantitative analysis, but qualitatively, the few that remained docked failed to fuse following Ca2+-addition. This implies that both C2B binding to Ca2+ and SNAREs are required for the Ca2+-activation, but the ability to form oligomers or Syt1-Cpx-SNARE tripartite interface are not crucial for the Ca2+-triggered reversal of the Syt1 clamp. The average values and standard deviations from four independent experiments are shown for each condition. In total,~250 vesicles were analyzed for each condition.