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This self-assembling proteinaceous organelle uses a polyhedral shell constructed by hundreds of shell protein paralogs to encapsulate the key CO2-fixing enzymes Rubisco and carbonic anhydrase. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding carboxysome formation and overall functionality. Here, we employed cryoelectron tomography and subtomogram averaging to delineate the 3D packaging of Rubiscos within \u03b2-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the \u03b2-carboxysome lumen. We also detected Rubisco binding with the scaffolding protein CcmM in \u03b2-carboxysomes, which is instrumental for Rubisco encapsulation and \u03b2-carboxysome assembly. Using Quantification conCATamer-based quantitative MS, we determined the absolute stoichiometric composition of the entire \u03b2-carboxysome. This study provides insights into the assembly principles and structural variation of \u03b2-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.<\/jats:p>","DOI":"10.1093\/plphys\/kiae665","type":"journal-article","created":{"date-parts":[[2024,12,16]],"date-time":"2024-12-16T18:39:20Z","timestamp":1734374360000},"source":"Crossref","is-referenced-by-count":25,"title":["Rubisco packaging and stoichiometric composition of the native \u03b2-carboxysome in <i>Synechococcus elongatus<\/i> PCC7942"],"prefix":"10.1093","volume":"197","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2175-7905","authenticated-orcid":false,"given":"Yaqi","family":"Sun","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3067-9520","authenticated-orcid":false,"given":"Yuewen","family":"Sheng","sequence":"additional","affiliation":[{"name":"Diamond Light Source , Harwell Science and Innovation Campus, Didcot OX11 0DE ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Ni","sequence":"additional","affiliation":[{"name":"Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford , Oxford OX3 7BN ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-9499-8978","authenticated-orcid":false,"given":"Xingwu","family":"Ge","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1942-6306","authenticated-orcid":false,"given":"Joscelyn","family":"Sarsby","sequence":"additional","affiliation":[{"name":"Centre for Proteome Research, Institute of Systems, Molecular and Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip J","family":"Brownridge","sequence":"additional","affiliation":[{"name":"Centre for Proteome Research, Institute of Systems, Molecular and Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9141-860X","authenticated-orcid":false,"given":"Kang","family":"Li","sequence":"additional","affiliation":[{"name":"College of Marine Life Sciences, and Frontiers Science Center for Deep Ocean Multispheres and Earth System, Ocean University of China , Qingdao 266003 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathan","family":"Hardenbrook","sequence":"additional","affiliation":[{"name":"Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford , Oxford OX3 7BN ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gregory F","family":"Dykes","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nichola","family":"Rockliffe","sequence":"additional","affiliation":[{"name":"Faculty of Health & Life Sciences, GeneMill, University of Liverpool , Crown Street, Liverpool L69 7ZB 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,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8884-4819","authenticated-orcid":false,"given":"Lu-Ning","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool , Crown Street, Liverpool L69 7ZB ,","place":["UK"]},{"name":"College of Marine Life Sciences, and Frontiers Science Center for Deep Ocean Multispheres and Earth System, Ocean University of China , Qingdao 266003 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2024,12,16]]},"reference":[{"issue":"5513","key":"2025040706382403700_kiae665-B1","doi-asserted-by":"publisher","first-page":"2594","DOI":"10.1126\/science.1055071","article-title":"Biospheric primary production during an ENSO 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