Human cells consist of a complex hierarchy of components, many of which remain unexplored1,2. Here we construct a global map of human subcellular architecture through joint measurement of biophysical interactions and immunofluorescence images for over 5,100 proteins in U2OS osteosarcoma cells. Self-supervised multimodal data integration resolves 275 molecular assemblies spanning the range of 10−8 to 10−5 m, which we validate systematically using whole-cell size-exclusion chromatography and annotate using large language models3. We explore key applications in structural biology, yielding structures for 111 heterodimeric complexes and an expanded Rag–Ragulator assembly. The map assigns unexpected functions to 975 proteins, including roles for C18orf21 in RNA processing and DPP9 in interferon signalling, and identifies assemblies with multiple localizations or cell type specificity. It decodes paediatric cancer genomes4, identifying 21 recurrently mutated assemblies and implicating 102 validated new cancer proteins. The associated Cell Visualization Portal and Mapping Toolkit provide a reference platform for structural and functional cell biology.
