@article {Evans2022.02.18.481072, author = {Sasha L. Evans and Monsour M. J. Al-Hazeem and Daniel Mann and Nicolas Smetacek and Andrew J. Beavil and Yaqi Sun and Taiyu Chen and Gregory F. Dykes and Lu-Ning Liu and Julien R. C. Bergeron}, title = {Single-particle cryo-EM analysis of the shell architecture and internal organization of an intact α-carboxysome}, elocation-id = {2022.02.18.481072}, year = {2022}, doi = {10.1101/2022.02.18.481072}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Carboxysomes are proteaceous bacterial microcompartments (BMCs) that sequester the key enzymes for carbon fixation in cyanobacteria and some proteobacteria. They consist of a virus-like icosahedral shell, encapsulating carbonic anhydrase and ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which catalyses the dehydration of bicarbonate into CO2, the first step of the Calvin{\textendash}Benson{\textendash}Bassham cycle. Despite their significance in carbon fixation and great bioengineering potentials, the structural characterization of native carboxysomes, including the shell and the internal organization, is currently limited to low-resolution tomography studies. Notably, the degree of heterogeneity of the shell, and the internal arrangement of enzymes, remain poorly understood. Here, we report the structural characterization of a native α-carboxysome from a marine cyanobacterium by single-particle cryo-EM. We determine the structure of RuBisCO enzyme at 2.9 {\r A} resolution. In addition, we obtain low-resolution maps of the icosahedral protein shell and the concentric interior organisation. In combination with artificial intelligence (AI)-driven modelling approaches, we exploited these maps to propose a complete atomic model of an intact carboxysome. This study provides insight into carboxysome structure and protein-protein interactions involved in carboxysome assembly. Advanced knowledge about carboxysome architecture and structural plasticity is critical for not only a better understanding of biological carbon fixation mechanism but also repurposing carboxysomes in synthetic biology for biotechnological applications.Competing Interest StatementThe authors have declared no competing interest.}, URL = {https://www.biorxiv.org/content/early/2022/02/18/2022.02.18.481072}, eprint = {https://www.biorxiv.org/content/early/2022/02/18/2022.02.18.481072.full.pdf}, journal = {bioRxiv} }