Roughly 20% of bacteria employ bacterial microcompartments (BMCs) to sequester enzymatic processes. In these structures, a cargo of enzymes and accessory proteins is encased within a semi-permeable protein shell that permits passage of substrates and products but restricts movement of intermediates. In addition to their importance as a component of many bacterial species’ metabolisms, BMCs have become a target of protein engineering due to their potential for enclosing cargos of choice for alternative pharmacological and biotechnological applications.<br/><br/>Despite the importance of BMCs in prokaryotic biology and bioengineering, structural heterogeneity has prevented a complete understanding of the architecture, ultrastructure, and spatial organization of these cellular compartments. We recently used cryo-electron tomography to image intact alpha-carboxysomes, a model BMC responsible for carbon fixation in many cyanobacteria and chemoautotrophs. Using a modified subtomogram averaging pipeline designed to prioritize information for ultrastructure analysis, we solved a 4.5 A in situ Rubisco structure that also defines location and orientation of ~99% of the Rubisco enzymes within the carboxysomes. We determined that the Rubisco forms a low-periodicity lattice with a symmetry mismatch between the enzyme and its assembly, and calculated concentration dependencies for ultrastructure formation. Here, we report new data on the impact of chemical environment on Rubisco lattice formation and function, as well as recent efforts to characterize additional protein components in the carboxysome. These data offer new insights into BMC organization and assembly as well as Rubisco's propensity for polymerization, and will provide potential tools for future bioengineering of BMCs.

Characterization of enzyme scaffolding inside the alpha-carboxysome by cryo-electron tomography

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