%0 Journal Article %A Sasha L. Evans %A Monsour M. J. Al-Hazeem %A Daniel Mann %A Nicolas Smetacek %A Andrew J. Beavil %A Yaqi Sun %A Taiyu Chen %A Gregory F. Dykes %A Lu-Ning Liu %A Julien R. C. Bergeron %T Single-particle cryo-EM analysis of the shell architecture and internal organization of an intact α-carboxysome %D 2022 %R 10.1101/2022.02.18.481072 %J bioRxiv %P 2022.02.18.481072 %X 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–Benson–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 Å 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. %U https://www.biorxiv.org/content/biorxiv/early/2022/02/18/2022.02.18.481072.full.pdf