Single-particle cryo-EM analysis of the shell architecture and internal organization of an intact α-carboxysome

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 -carboxysome from a marine cyanobacterium by single-particle cryo-EM. We determine the structure of RuBisCO enzyme at 2.9 [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.