Updated project metadata. Mitochondrial complex I is a redox-driven proton pump that generates most of the proton-motive force powering oxidative phosphorylation and ATP synthesis in eukaryotes. We report the structure of complex I from the thermophilic eukaryote Chaetomium thermophilum, determined by electron cryo-microscopy to 2.4 A resolution in the open and closed conformation. Complex I has two arms, the peripheral and membrane arm, forming an L-shape. The two conformations differ in the relative position of the two arms. The open-to-closed transition is accompanied by substantial conformational changes in the Q-binding cavity and the E-channel, and by the formation of an aqueous connection between the E-channel and an extensive aqueous passage inside the membrane arm. The observed similarities provide strong support for a conserved, common mechanism that applies across all species from fungi to mammals. Furthermore, the complex is inhibited by the detergent DDM, which binds reversibly to two sites in the Q-binding cavity.