{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T04:53:38Z","timestamp":1790916818149,"version":"4.1.0"},"posted":{"date-parts":[[2026,7,28]]},"group-title":"Biochemistry","reference-count":25,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T00:00:00Z","timestamp":1785196800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["GM124847"],"award-info":[{"award-number":["GM124847"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"award":["U24 GM129539"],"award-info":[{"award-number":["U24 GM129539"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R24 GM154192"],"award-info":[{"award-number":["R24 GM154192"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"award":["T32GM138826"],"award-info":[{"award-number":["T32GM138826"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"award":["SF349247"],"award-info":[{"award-number":["SF349247"]}],"id":[{"id":"https:\/\/ror.org\/01cmst727","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,30]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>Cobalamin-dependent methionine synthase (MetH) is a highly dynamic enzyme that requires large-scale domain rearrangements to facilitate turnover and reactivation. Despite significant biochemical and structural efforts, a full picture of the conformational changes underlying the transition between MetH turnover and reactivation has remained elusive. Here, we leverage state-of-the-art cryo-electron microscopy (cryo-EM) methods to reanalyze an old dataset and gain new insights into these changes. By utilizing a neural-network particle picker, performing careful classification of particles, and incorporating high-resolution information to determine initial particle orientations, we uncovered and reconstructed a minor population of reactivation-state MetH (\u223c4.5-5 \u00c5 resolution) from a dataset that is dominated by resting-state MetH. This structure represents the first fully intact structure of the enzyme with a physiological cobalamin cofactor bound. These findings highlight the potential for improved cryo-EM methodology to uncover previously hidden details about enzyme function and deepen our understanding of structural ensembles.<\/jats:p>","DOI":"10.64898\/2026.07.27.741092","type":"posted-content","created":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T12:20:25Z","timestamp":1785241225000},"source":"Crossref","is-referenced-by-count":0,"title":["Recovery of a minor cryo-EM particle population reveals conformational equilibria linking cofactor loading, turnover, and reactivation in methionine synthase"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2933-0030","authenticated-orcid":false,"given":"Haoyue","family":"Wang","sequence":"first","affiliation":[{"name":"Field of Biophysics, Cornell University, Ithaca, NY 14853, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4559-2049","authenticated-orcid":false,"given":"Maxwell B.","family":"Watkins","sequence":"additional","affiliation":[{"name":"Biophysics Collaborative Access Team (BioCAT), Department of Biology, Illinois Institute of Technology, Chicago, IL 60616, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8317-6073","authenticated-orcid":false,"given":"Pasa","family":"Suksmith","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7062-1644","authenticated-orcid":false,"given":"Nozomi","family":"Ando","sequence":"additional","affiliation":[{"name":"Field of Biophysics, Cornell University, Ithaca, NY 14853, USA"},{"name":"Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026100121000731000_2026.07.27.741092v2.1","doi-asserted-by":"publisher","DOI":"10.1021\/bi961693s"},{"key":"2026100121000731000_2026.07.27.741092v2.2","doi-asserted-by":"publisher","DOI":"10.1021\/bi9730893"},{"key":"2026100121000731000_2026.07.27.741092v2.3","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1133218100"},{"key":"2026100121000731000_2026.07.27.741092v2.4","doi-asserted-by":"publisher","DOI":"10.1126\/science.7992050"},{"key":"2026100121000731000_2026.07.27.741092v2.5","doi-asserted-by":"publisher","DOI":"10.1016\/S0969-2126(96)00135-9"},{"key":"2026100121000731000_2026.07.27.741092v2.6","doi-asserted-by":"publisher","DOI":"10.1038\/nsb738"},{"key":"2026100121000731000_2026.07.27.741092v2.7","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0308082100"},{"key":"2026100121000731000_2026.07.27.741092v2.8","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0709960105"},{"key":"2026100121000731000_2026.07.27.741092v2.9","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0800329105"},{"key":"2026100121000731000_2026.07.27.741092v2.10","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0906132106"},{"key":"2026100121000731000_2026.07.27.741092v2.11","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1107\/S2059798317018290","article-title":"The folate-binding module of Thermus thermophilus cobalamin-dependent methionine synthase displays a distinct variation of the classical TIM barrel: a TIM barrel with a \u2018twist","volume":"74","year":"2018","journal-title":"Acta Crystallogr D Struct Biol"},{"key":"2026100121000731000_2026.07.27.741092v2.12","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2302531120"},{"key":"2026100121000731000_2026.07.27.741092v2.13","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-42037-4"},{"key":"2026100121000731000_2026.07.27.741092v2.14","unstructured":"Mendoza, J. et al. Structural Snapshots of B12-Dependent Methionine Synthase\u2019s Catalytic Conformations. bioRxiv (2024)."},{"key":"2026100121000731000_2026.07.27.741092v2.15","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-026-72899-3"},{"key":"2026100121000731000_2026.07.27.741092v2.16","doi-asserted-by":"publisher","DOI":"10.1021\/bi952389m"},{"key":"2026100121000731000_2026.07.27.741092v2.17","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4169"},{"key":"2026100121000731000_2026.07.27.741092v2.18","doi-asserted-by":"publisher","DOI":"10.1107\/S2052252520000081"},{"key":"2026100121000731000_2026.07.27.741092v2.19","doi-asserted-by":"publisher","DOI":"10.1107\/S205225251801463X"},{"key":"2026100121000731000_2026.07.27.741092v2.20","doi-asserted-by":"crossref","unstructured":"Kim, K. , Li, H. & Clarke, O. B. High-resolution ab initio reconstruction enables cryo-EM structure determination of small particles. bioRxiv (2025).","DOI":"10.1101\/2025.09.08.674935"},{"key":"2026100121000731000_2026.07.27.741092v2.21","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-020-00990-8"},{"key":"2026100121000731000_2026.07.27.741092v2.22","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-019-0575-8"},{"key":"2026100121000731000_2026.07.27.741092v2.23","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2007.05.022"},{"key":"2026100121000731000_2026.07.27.741092v2.24","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.21303"},{"key":"2026100121000731000_2026.07.27.741092v2.25","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkv314"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.07.27.741092","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T04:00:13Z","timestamp":1790913613000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.07.27.741092"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,28]]},"references-count":25,"URL":"https:\/\/doi.org\/10.64898\/2026.07.27.741092","relation":{},"subject":[],"published":{"date-parts":[[2026,7,28]]},"subtype":"preprint"}}