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This process is inhibited by kasugamycin, edeine and GE81112, however, their mechanisms of action have not been fully elucidated. Here we present cryo-electron microscopy structures of 30S initiation intermediate complexes formed in the presence of kasugamycin, edeine and GE81112 at resolutions of 2.0-2.9\u2009\u00c5. The structures reveal that all three antibiotics bind within the E-site of the 30S and preclude 30S initiation complex formation. While kasugamycin and edeine affect early steps of 30S pre-initiation complex formation, GE81112 stalls pre-initiation complex formation at a further step by allowing start codon recognition, but impeding IF3 departure. Collectively, our work highlights how chemically distinct compounds binding at a conserved site on the 30S can interfere with translation initiation in a\u00a0unique manner.<\/jats:p>","DOI":"10.1038\/s41467-025-57731-8","type":"journal-article","created":{"date-parts":[[2025,3,12]],"date-time":"2025-03-12T12:04:00Z","timestamp":1741781040000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["The translation inhibitors kasugamycin, edeine and GE81112 target distinct steps during 30S initiation complex formation"],"prefix":"10.1038","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3533-090X","authenticated-orcid":false,"given":"Haaris A.","family":"Safdari","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martino","family":"Morici","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3861-3083","authenticated-orcid":false,"given":"Ana","family":"Sanchez-Castro","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea","family":"Dallap\u00e8","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8335-3239","authenticated-orcid":false,"given":"Helge","family":"Paternoga","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna Maria","family":"Giuliodori","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3928-2781","authenticated-orcid":false,"given":"Attilio","family":"Fabbretti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6679-5473","authenticated-orcid":false,"given":"Pohl","family":"Mil\u00f3n","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3816-3828","authenticated-orcid":false,"given":"Daniel N.","family":"Wilson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,3,12]]},"reference":[{"key":"57731_CR1","doi-asserted-by":"publisher","first-page":"712","DOI":"10.1016\/j.molcel.2008.04.014","volume":"30","author":"P Milon","year":"2008","unstructured":"Milon, P., Konevega, A. 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