Project Summary/Abstract Bacteria lack organelles, yet some enzymatic pathways generate intermediates which are either susceptible to loss or which could be toxic to the cell if released into the cytosol. Roughly 20% of bacteria encase the potentially dangerous or inefficient components of these pathways inside bacterial microcompartments (BMCs). BMCs have been characterized in a number of species, and serve a variety of roles from carbon fixation to small molecule metabolism. They typically contain two or more enzymes, a variable number of accessory and scaffolding proteins, and a compact protein shell that is selectively permeable to small molecules without the aid of a lipid membrane. The shell proteins and the heterogeneous composition of the shell are conserved across all BMCs of all functions characterized thus far. Because BMCs can allow bacteria to live in hostile environments, they have broad implications for human health, ecology and infectious disease. BMCs have also become a target of protein engineering due to their potential for enclosing cargos of choice for alternative pharmacological and biotechnological applications, as well as for their basic value as a critical component of many species’ metabolism. Despite their importance, structural heterogeneity has prevented a complete understanding of architecture, ultrastructure, and spatial organization of both the shell proteins and the cargo. The research proposed here seeks to characterize the structure and organization of carboxysomes, a model BMC responsible for carbon fixation in cyanobacteria. High-resolution cryo-electron tomography and sub-tomogram averaging will be used to determine cargo organization and shell ultrastructure in vitro and in vivo, preserving and characterizing the conserved heterogeneity of the structures. In addition to providing new insights into BMC biology and its conserved complexity, this research will also generate new analysis methods that can be applied to many complicated BMC and viral systems.