This PDF file includes:

• Supplementary Text
• fig. S1. Line shape of intensity traces from single DCV fusion events.
• fig. S2. Ensemble lipid mixing of DCVs with reconstituted proteoliposomes containing syntaxin-1a (residues 183 to 288) (lipid/protein = 500) with a lipid composition of bPC:bPE:bPS:Chol:PI:PI(4,5)P₂:Rh-DOPE:NBD-DOPE (23.5:23.5:15:30:4:1:1.5:1.5) at increasing [Ca²⁺].
• fig. S3. Single DCV fusion response.
• fig. S4. Quantification of shRNA-mediated knockdowns.
• fig. S5. DCV fusion kinetics of synaptotagmin and CAPS knockdowns.
• fig. S6. Western blots of complexin-1/2, Munc18, and Munc13 in fractions generated during DCV purification.
• fig. S7. Controls to determine the effects of Munc18 and complexin-1 on docking to full-length syntaxin-1a (residues 1 to 288) and SNAP-25 and distinguish which of these conditions are inhibited by the soluble domain of synaptobrevin-2 (residues 1 to 96).
• fig. S8. Complexin-1 inhibits fusion to planar supported bilayers containing syntaxin-1a (residues 1 to 288):SNAP-25 in the presence of 0.5 μM Munc18 in the absence of calcium, whereas there is no effect on DCV docking.
• fig. S9. Fusion probability as a function of calcium for fusion of DCVs that were docked to planar supported bilayers with syntaxin-1a (residues 1 to 288):SNAP-25 in the presence of Munc18 and complexin-1 upon triggering with calcium.
• table S1. Event statistics for DCV docking and fusion to planar supported bilayers with syntaxin-1a (residues 183 to 288):SNAP-25 (lipid/protein = 3000), syntaxin-1a (residues 183 to 288) (lipid/protein = 3000), dodecylated SNAP-25 (lipid/protein = 3000), no protein, or syntaxin-1a (residues 183 to 288):SNAP-25
• (lipid/protein = 3000) incubated with 2 μM synaptobevin-2 (residues 1 to 96) inhibitor peptide.
• table S2. Event statistics for DCV docking and fusion to planar supported bilayers with syntaxin-1a (residues 183 to 288):SNAP-25 (lipid/protein = 3000) with increasing concentration of divalent metals (Ca²⁺ and Mg²⁺).
• table S3. Fit values of a parallel reaction model [N(t) = N(1 − e^−kt)^m, where N is the fusion probability, k is the rate, and m is the number of parallel reactions occurring] for the cumulative distribution function of delay times between docking and fusion for single-particle DCV events with different concentrations of divalent metals.
• table S4. Fits of ensemble lipid mixing data shown in fig. S3.
• table S5. Event statistics for docking and fusion of DCVs to planar supported bilayers with 5% PI(4,5)P₂ [bPC:bPE:bPS:Chol:PI(4,5)P₂ (25:25:15:30:5)], 3% PI(4,5)P₂ [bPC:bPE:bPS:Chol:PI:PI(4,5)P₂ (25:25:15:30:2:3)], 1% PI(4,5)P₂ [bPC:bPE:bPS:Chol:PI:PI(4,5)P₂ (25:25:15:30:4:1)], 0% PI(4,5)P₂ [bPC:bPE:bPS:Chol:PI (25:25:15:30:5)], and no charged lipids [bPC:bPE:Chol (35:35:30)] in the presence of 100 μM EDTA or 100 μM Ca²⁺.
• table S6. Event statistics for docking and fusion of DCVs to planar supported bilayers with 5% PI(4,5)P₂ [bPC:bPE:bPS:Chol:PI(4,5)P₂ (25:25:15:30:5)], 3% PI(4,5)P₂ [bPC:bPE:bPS:Chol:PI:PI(4,5)P₂ (25:25:15:30:2:3)], 1% PI(4,5)P₂ [bPC:bPE:bPS:Chol:PI:PI(4,5)P₂ (25:25:15:30:4:1)], 0% PI(4,5)P₂ [bPC:bPE:bPS:Chol:PI (25:25:15:30:5)], and no charged lipids [bPC:bPE:Chol (35:35:30)] in the presence of 100 μM EDTA or 100 μM Ca²⁺.
• table S7. Event statistics for DCV docking and fusion to planar supported bilayers with syntaxin-1a (residues 183 to 288):SNAP-25 (lipid/protein = 3000) with wild-type DCVs, DCVs inhibited with antibodies for either syt or CAPS, or DCVs that have had syt or CAPS knocked down using shRNA before purification.
• table S8. Fit values of a parallel reaction model [N(t) = N(1 − e^−kt)^m, where N is the fusion probability, k is the rate, and m is the number of parallel reactions occurring] for the cumulative distribution function of delay times between docking and fusion for single-particle DCV events for conditions described in Fig. 2.
• table S9. Event statistics for DCV docking and fusion to planar supported bilayers containing either syntaxin-1a (residues 183 to 288) or syntaxin-1a (residues 1 to 288) with SNAP-25 (lipid/protein = 3000), with the addition of complexin-1 and/or Munc18 in the presence of 100 μM EDTA or 100 μM Ca2+.
• table S10. Fit values of a parallel reaction model [N(1 − e^−kt)^m, where N is the fusion probability, k is the rate, and m is the number of parallel reactions occurring] for the cumulative distribution function of delay times between docking and fusion for single-particle DCV events for conditions described in Fig. 3.
• table S11. Event statistics of triggered fusion of DCVs at different calcium concentrations from data shown in Fig. 4B. table S12. Event statistics of spontaneous fusion of DCVs docked in triggering conditions with planar supported bilayers of different lipid compositions.
• table S13. Event statistics of triggered fusion of DCVs with planar supported bilayers of different lipid compositions.
• table S14. Event statistics of spontaneous fusion of DCVs docked in triggering conditions with knockdowns of syt-1/9 or CAPS with subsequent recoveries.
• table S15. Event statistics of triggered fusion of DCVs with knockdowns of syt-1/9 or CAPS and subsequent recoveries.
• table S16. Table of primers described in the “Plasmids and shRNA constructs” section of Materials and Methods.
• table S17. Event statistics for DCV docking and fusion to planar supported bilayers with the indicated combination of proteins syntaxin-1a (residues 183 to 288), syntaxin-1a (residues 1 to 288), and SNAP-25 (lipid/protein = 3000) incubated, as indicated, with Munc18 (0.5 μM), complexin-1 (2 μM), and synaptobrevin-2 (residues 1 to 96) inhibitor peptide.
• table S18. Event statistics for DCV docking and fusion to planar supported bilayers containing syntaxin-1a (residues 1 to 288):SNAP-25, 0.5 μM Munc18, and indicated amounts of complexin-1.
• References (70–74)

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