Abstract Cells regulate growth and function through a hierarchical structure of subcellular protein assemblies, in which alterations can result in cellular dysfunction leading to disease such as cancer. Much of this structure remains uncharted, resulting in recent efforts to map subcellular organization at different physical scales. Here, we report a global architectural map of human cancer cell protein assemblies spanning 10−9 to 10−5 nm, based on integration of near-proteome-wide affinity purification mass spectrometry-based protein interactions and immunofluorescent imaging in U-2 OS osteosarcoma cancer cells. The U-2 OS multi-scale integrated cell map places >5000 proteins into 270 distinct subcellular protein assemblies across biological scales, representing approximately half of expressed proteins. There are known organelles and protein complexes recovered in the map, as well as 152 putative assemblies, such as a putative interferon signaling complex consisting of a serine protease and STAT transcription factors. The map also incorporates 128 previously uncharacterized proteins into protein assemblies, such as C18orf21 association with a canonical RNAse complex. Protein subsystems in the U-2 OS map were evaluated for the potential for downstream integrative structural modeling, where available structural data (e.g. crosslinking, Protein Data Bank structures, AlphaFold predictions) are combined in a Bayesian framework to model the physical structures of protein complexes. Using this framework, we created an integrated structural model of new proteins interacting with the Rag-Ragulator complex, which regulates MAPK and mTOR signaling. In summary, the global proteome architecture map provides a resource for cellular biology discovery and the systematic determination of protein functions and structures. Citation Format: Leah V. Schaffer, Mengzhou Hu, Edward L. Huttlin, Gege Qian, Abantika Pal, Neelesh Soni, Andrew Latham, Laura Pontano Vaites, Trang Le, Yue Qin, Christopher Churas, Dexter Pratt, Ignacia Echeverria, Andrej Sali, J Wade Harper, Steven P. Gygi, Emma Lundberg, Trey Ideker. A global multiscale map of protein assemblies from integration of protein interactions and images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2348.