Imaging of donor/acceptor interface morphologies by cryo-EM before and after Ca2+ addition. (A–F) Cryo-EM images of mixtures of donor (synaptobrevin and synaptotagmin 1) and acceptor (syntaxin and SNAP-25) vesicles before (A–C) and approximately 35 s after (D–F) 500 μM Ca2+ addition (panels B, C, E, and F are close-up views). Vesicles were imaged in the holes of the substrate carbon film, visible as the darker areas in the image, in conditions that clearly show the lipid bilayers (‘Materials and methods’). The particular images shown in this figure were selected out of total of 16 (before Ca2+ addition) and 21 (after Ca2+ addition) EM micrographs, respectively, with emphasis on showing point contacts before Ca2+ addition and extended interfaces after Ca2+ addition in order to illustrate the variety of these particular states. Arrows indicate interfaces between vesicles that are approximately perpendicular to the direction of the projection (see ‘Materials and methods’ for definitions of all contact and interface types). Large black arrow: point contact (representative close-up in B), small black/white arrow: hemifusion diaphragm (representative close-ups in C and E), and large white arrow: extended close contact (representative close-up in F). Scale bars in A and D are 100 nm, and 20 nm in B, C, E, and F. (G) Distribution of vesicle sizes before and after Ca2+ addition. Vesicle diameters were calculated from all cryo-EM images in both conditions (‘Materials and methods’). (H) Bar graph of the percentage of various vesicle interfaces, that is, point contacts, hemifusion diaphragms, and extended close contacts (including a few instances of mixed, i.e., extended/hemifused, interfaces), normalized with respect to the total number of interfaces observed before and after addition of 500 μM Ca2+, respectively (‘Materials and methods’). In addition to the changes in the distribution of vesicle interfaces upon Ca2+ addition, some amount of complete fusion between vesicles occurred as indicated by the shift of the diameter distribution in panel G towards larger values. Source files of all cryo-EM micrographs used for the quantitative analysis are available in the Figure 2—source data 1.