This study reveals the nanoscale architecture of synaptic vesicles and scaffolding complexes in synapses using cryo-electron tomography (cryo-ET) combined with focused-ion beam (FIB) milling. The research shows that synaptic vesicles are not preferentially aligned with scaffolding nanoclusters in the presynaptic active zone (AZ) or postsynaptic density (PSD), suggesting that the spatial relationship between scaffolding nanoclusters and synaptic vesicles influences synaptic strength. Cryo-ET images under near-native conditions reveal detailed ultrastructural features of synapses, including supramolecular nanoclusters of AZ and PSD proteins, and synaptic vesicles linked to the membrane by peripheral protein densities. These densities are consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using tomograms suggest that clustering AMPA receptors within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. The study highlights the importance of nanoscale topography in regulating synaptic signal transduction and plasticity. The findings support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites. The research provides new insights into the structural and functional organization of synapses, with implications for understanding synaptic transmission and plasticity.This study reveals the nanoscale architecture of synaptic vesicles and scaffolding complexes in synapses using cryo-electron tomography (cryo-ET) combined with focused-ion beam (FIB) milling. The research shows that synaptic vesicles are not preferentially aligned with scaffolding nanoclusters in the presynaptic active zone (AZ) or postsynaptic density (PSD), suggesting that the spatial relationship between scaffolding nanoclusters and synaptic vesicles influences synaptic strength. Cryo-ET images under near-native conditions reveal detailed ultrastructural features of synapses, including supramolecular nanoclusters of AZ and PSD proteins, and synaptic vesicles linked to the membrane by peripheral protein densities. These densities are consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using tomograms suggest that clustering AMPA receptors within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. The study highlights the importance of nanoscale topography in regulating synaptic signal transduction and plasticity. The findings support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites. The research provides new insights into the structural and functional organization of synapses, with implications for understanding synaptic transmission and plasticity.