a, Table of results from assays that tested the ability of plasmids encoding E. coli LptF variants to complement a ΔlptF strain. b, To assess whether the LptF double-cysteine mutant was being expressed and translated properly, we measured LptF–Flag levels by anti-Flag western blot in whole-cell lysates from merodiploid E. coli strains containing either pBAD18-LptF-Flag or pBAD18-LptF(S157C/I234C)-Flag. c, Disc diffusion assays were carried out to assess the outer-membrane permeability (relative to wild type) of E. coli strains expressing only plasmid-encoded LptF variants. Values denote the diameter (in mm) of regions with no growth, and values in parentheses denote regions of inhibited growth. Discs were 6 mm in diameter. d, Disc diffusion assays were performed as in c, except that experiments were performed in merodiploid strains with wild-type chromosomal copies of lptFG. Alleles are listed as follows: plasmid-encoded lptFG alleles, a solidus and then the chromosome-encoded lptFG alleles. e, Coomassie-stained samples of purified LptB 2 FGC used in the LPS release assay: (1) LptF–Flag; (2) LptF(S157C)–Flag; (3) LptF(I234C)–Flag; and (4) LptF(S157C/I234C)–Flag. LptC contained a C-terminal thrombin-cleavable His 7 tag used for nickel-affinity chromatography, which was cleaved before reconstitution into liposomes. f, The ATPase activity of E. coli LptB 2 FGC reconstituted into liposomes was assessed by measuring the rate of phosphate release over time. Data are mean ± s.d. of results from three technical replicates, each of two biological replicates, except two replicates that were performed with one LptB(E163Q) 2 FGC sample. Data in b and e are representative of data from three biological replicates. Data in c and d are mean ± s.d. calculated from three independent experiments.