Extended Data Fig. 6: LPS enters the cavity of LptB₂FGC via LptG TM1, LptF TM5 and the LptC transmembrane helix in a nucleotide-independent manner.

a, Western blots from Fig. 3b are shown alongside blots from additional in vivo photocrosslinking experiments comparing LPS crosslinking in wild-type and LptB(E163Q) backgrounds. b, In vitro reconstitution (as in Extended Data Fig. 1b) of LptB₂FGC, LptB₂FGC(M19pBPA), LptB₂FGC(G21pBPA) and LptB₂FGC(F78pBPA) shows that the variants with amber codons incorporated in the LptC transmembrane helix release LPS to LptA(I36pBPA) as well as wild type. c, In vitro photocrosslinking of LptB₂FGC pBPA variants reconstituted into proteoliposomes with LPS, as in Fig. 3d, comparing complexes containing LptB(E163Q) to those containing wild-type LptB, and the effects of AMPPNP relative to ATP. d–f, Additional crosslinking experiments with pBPA incorporated at other positions in the transmembrane helix of LptC, LptF TM1 and TM5, and LptG TM1 and TM5. g, h, Ribbon diagrams of E. cloacae LptB₂FGC, with views of the two potential gates in the LptFGC transmembrane helices; LptG (teal), LptC (pink), and LptF (green). Residues of interest are shown as sticks. Blots for LptG(S30pBPA) are shown in Extended Data Fig. 7b. Blots shown in a–f are representative of data from three biological replicates.