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Rubisco has low selectivity for its substrate, CO\n                  <jats:sub>2<\/jats:sub>\n                  , and has a slow enzymatic turnover rate, resulting in an inefficient metabolic pathway. Within the \u03b1-CB, Rubisco has been observed at a range of concentrations and with either a liquid-like assembly or a pseudo-lattice of polymerized fibrils. The biophysical origins of the fibril ultrastructure organization are unclear; however, it is only observed inside \u03b1-CBs. Quantitative knowledge of the binding constants and energies for assembly and maintenance of these fibrils is critical for understanding this organization and Rubisco regulation, but quantitative methods for\n                  <jats:italic>in situ<\/jats:italic>\n                  analysis of Rubisco polymerization have been lacking. Here, we present an approach to convert tomography-derived \u03b1-CB volumes and Rubisco particle positions into polymerization binding curves. We used this procedure to determine the Rubisco polymerization constants, including the nucleus size (\n                  <jats:italic>n<\/jats:italic>\n                  ) and equilibrium polymerization constant (\n                  <jats:italic>K<\/jats:italic>\n                  <jats:sub>\n                    <jats:italic>pol<\/jats:italic>\n                  <\/jats:sub>\n                  ). The adopted modeling approach is consistent with\n                  <jats:italic>in situ<\/jats:italic>\n                  constraints, such as concentration-dependent binding interactions and confinement. This approach offers a powerful tool to evaluate both\n                  <jats:italic>in vitro<\/jats:italic>\n                  and potentially\n                  <jats:italic>in vivo<\/jats:italic>\n                  biomolecular interactions, both of Rubisco and of other proteins and polymers suitable for analysis by cryo-electron tomography.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>Cryogenic electron tomography (cryoET) is a powerful method to resolve structures of proteins in their native environment at subnanometer-level resolution. Because tomography data retains spatial relationships of all particles, it intrinsically contains information about component (e.g., protein) binding interactions. Here, we use Rubisco polymerization in \u03b1-carboxysomes as a model system to demonstrate that quantitative, biochemical binding analysis is possible with cryoET.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.03.20.713215","type":"posted-content","created":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T05:10:27Z","timestamp":1774329027000},"source":"Crossref","is-referenced-by-count":0,"title":["Using Cryogenic Electron Tomography (cryoET) to Determine Rubisco Polymerization Constants in \u03b1-Carboxysomes"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-3638-9586","authenticated-orcid":false,"given":"Wenxiang","family":"Cao","sequence":"first","affiliation":[{"name":"Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5692-6046","authenticated-orcid":false,"given":"Kristy","family":"Rochon","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Purdue University, West Lafayette, IN, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan H.","family":"Gray","sequence":"additional","affiliation":[{"name":"James Tarpo Jr. and Margaret Tarpo Department of Chemistry, Purdue University, West Lafayette, IN, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5716-9980","authenticated-orcid":false,"given":"Luke","family":"Oltrogge","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, United States"},{"name":"Howard Hughes Medical Institute, University of California; Berkeley, CA, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0042-2257","authenticated-orcid":false,"given":"David F.","family":"Savage","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, United States"},{"name":"Howard Hughes Medical Institute, University of California; Berkeley, CA, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4798-2892","authenticated-orcid":false,"given":"Enrique M.","family":"De La Cruz","sequence":"additional","affiliation":[{"name":"Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8073-6960","authenticated-orcid":false,"given":"Lauren Ann","family":"Metskas","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Purdue University, West Lafayette, IN, United States"},{"name":"James Tarpo Jr. and Margaret Tarpo Department of Chemistry, Purdue University, West Lafayette, IN, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041708050656000_2026.03.20.713215v3.1","doi-asserted-by":"publisher","DOI":"10.1021\/bi201284u"},{"key":"2026041708050656000_2026.03.20.713215v3.2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.75.103004.142710"},{"key":"2026041708050656000_2026.03.20.713215v3.3","doi-asserted-by":"publisher","DOI":"10.1016\/S0968-0004(98)01187-6"},{"key":"2026041708050656000_2026.03.20.713215v3.4","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-060614-034142"},{"key":"2026041708050656000_2026.03.20.713215v3.5","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E10-08-0683"},{"key":"2026041708050656000_2026.03.20.713215v3.6","doi-asserted-by":"publisher","DOI":"10.7554\/elife.57264"},{"key":"2026041708050656000_2026.03.20.713215v3.7","doi-asserted-by":"crossref","unstructured":"A. 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