Morphology of WT and RIM1α KO synapses by cryo-ET. In unstained, vitrified frozen-hydrated mammalian synapses, the presynaptic cytomatrix mainly consists of filaments shorter than 40 nm linking vesicles to each other (connectors) or to the AZ (tethers). (A, D and F) Tomographic slices of WT (A), RIM1α KO-altered (D), and RIM1α KO-aligned (F) synapses. mit, mitochondrion; PSD, postsynaptic density; SC, synaptic cleft; SV, synaptic vesicle. Bars, 100 nm. B, E, and G show corresponding 3D renderings of all vesicles within 250 nm from the AZ (left) and of the AZ and proximal vesicles seen from the cytoplasmic side (right). AZ (gray), synaptic vesicles (yellow), tethers (blue), connectors (red) are shown. For scale reference, mean vesicle diameter was 40.1 ± 5.4 nm (mean ± SD; no scale bars are shown because the image is rendered with 3D perspective). RIM1α KO-altered synapses showed reduced number of proximal vesicles and vesicle tethering to the AZ. (C) Magnified views of connectors (black arrowheads) and tethers (white arrowheads). Bars, 50 nm. Tomographic slices are 5.4 nm thick. Video 1, Video 2, and Video 3 are related.