Synaptic dysfunction is a key feature of SHANK-associated disorders such as autism spectrum disorder, schizophrenia, and Phelan-McDermid syndrome. Since detailed knowledge of their effect on synaptic nanostructure remains limited, we investigated such alterations in ex11|SH3 SHANK3-KO mice combining expansion (ExM) and STED microscopy. This enabled high-resolution imaging of mosaic-like arrangements formed by synaptic proteins. We found distinct shape-profiles as fingerprints of the murine postsynaptic scaffold across brain regions and genotypes, as well as alterations in the spatial and molecular organization of subsynaptic domains under SHANK3-deficient conditions. These results provide insights into synaptic nanostructure in situ and advance our understanding of molecular mechanisms underlying synaptic dysfunction in neuropsychiatric disorders. By combining whole-cell patch clamp recordings with post-hoc ExM, we now aim to analyze how synaptic profiles influence electrophysiological properties of single murine neurons.