Anton, Jana SJana SAntonIacovache, IoanIoanIacovacheBada Juarez, Juan FJuan FBada JuarezAbriata, Luciano ALuciano AAbriataPerrin, Louis WLouis WPerrinCao, ChanChanCaoMarcaida, Maria JMaria JMarcaidaZuber, BenoîtBenoîtZuber0000-0001-7725-5579Dal Peraro, MatteoMatteoDal Peraro2025-03-122025-03-122025-02-03https://boris-portal.unibe.ch/handle/20.500.12422/204707Jana S. Anton and Ioan Iacovache contributed equally to this work.Aerolysin is a β-pore-forming toxin produced by most Aeromonas bacteria, which has attracted large attention in the field of nanopore sensing due to its narrow and charged pore lumen. Structurally similar proteins, belonging to the aerolysin-like family, are present throughout all kingdoms of life, but very few of them have been structurally characterized in a lipid environment. Here, we present the first high-resolution atomic cryo-EM structures of aerolysin prepore and pore in a membrane-like environment. These structures allow the identification of key interactions, which are relevant for understanding the pore formation mechanism and for correctly positioning the pore β-barrel and its anchoring β-turn motif in the membrane. Moreover, we elucidate at high resolution the architecture of key pore mutations and precisely identify four constriction rings in the pore lumen that are highly relevant for nanopore sensing experiments.en600 - Technology::610 - Medicine & healtharticle10.48620/858773990053110.1021/jacs.4c14288