(E) Incubation scheme: GUVs (14 nmol lipid) containing preassembled t-SNAREs (14 pmol Syx1/SNAP-25) were mixed on ice with the indicated Munc18-1 constructs (90 pmol) and subsequently SUVs (2.5 nmol lipid) containing Synaptotagmin1 (3 pmol) and Synaptobrevin/VAMP2 (8 pmol) were added. The fusion reaction was started by warming up the liposomes to 37 °C. Calcium (100 µM final concentration) was added after 2 minutes. 