(A) Side view of the ∼8 Å resolution cryo-EM based electron density of the RNC:YidC complex, with the small subunit depicted in yellow, the large subunit in gray, P-site tRNA and nascent chain in green, YidC in red and the detergent micelle in blue. (B) As in A, but sliced through the ribosomal exit tunnel. (C) Validation of the active state model by disulphide crosslinking. RNCs carrying the mutant FOc(G23C) were reconstituted with the indicated single cysteine YidC mutants, oxidized, applied to a linear sucrose gradient and harvested from the 70S peak before SDS-PAGE and western blotting. Immunodetection was performed with antibodies raised against the HA-tag (located in the nascent chain inside the ribosomal exit tunnel) and anti-YidC antibodies. YidC, nascent chain-tRNA (NC-tRNA) and the expected crosslink product (NC-tRNA x YidC) are indicated. (D–F) Structural model of YidC during membrane protein insertion, viewed from two sides within the membrane (D and E) and from the cytoplasm (F). The detergent micelle was removed for clarity, the TM helix of FOc is depicted in magenta, and the disulphide crosslink between YidC and FOc with -SS-.