LAUSR

Significance Imaging the ultrastructure and molecular architecture of synapses is essential to understanding synaptic neurotransmission. Scaffolding proteins on both sides of the synapse form subsynaptic clusters that are aligned across the synapse. This is thought to concentrate the proteins required for presynaptic vesicle fusion across from postsynaptic receptors to mediate efficient neurotransmission. We used focused-ion beam milling and cryoelectron tomography to obtain 3D images of synapses under near-native conditions, allowing visualization of both synaptic vesicles and clustered scaffolding proteins. While scaffolding complexes are aligned across the synapse, membrane-proximal synaptic vesicles are offset from clustered scaffolds, suggesting a role for these geometric properties of synapses in determining the amplitude and variability of the synaptic response to vesicle fusion.