The study investigates the nanoscale architecture of synaptic vesicles and scaffolding complexes using cryo-electron tomography (cryo-ET) and focused-ion beam milling (FIB). The researchers found that while the postsynaptic density (PSD) and presynaptic active zone (AZ) form trans-synaptic "nanocolumns" to align proteins required for efficient neurotransmission, membrane-proximal synaptic vesicles are not preferentially aligned with these nanoclusters. Instead, these vesicles are linked to the membrane by peripheral protein densities, often resembling Munc13, and globular densities that bridge the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by the tomograms predict that clustering AMPA receptors within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. The findings support a model where synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.The study investigates the nanoscale architecture of synaptic vesicles and scaffolding complexes using cryo-electron tomography (cryo-ET) and focused-ion beam milling (FIB). The researchers found that while the postsynaptic density (PSD) and presynaptic active zone (AZ) form trans-synaptic "nanocolumns" to align proteins required for efficient neurotransmission, membrane-proximal synaptic vesicles are not preferentially aligned with these nanoclusters. Instead, these vesicles are linked to the membrane by peripheral protein densities, often resembling Munc13, and globular densities that bridge the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by the tomograms predict that clustering AMPA receptors within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. The findings support a model where synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.