Extended Data Fig. 9: Complementation and purification of LptF cysteine mutants.
From: Structural basis of unidirectional export of lipopolysaccharide to the cell surface

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 LptB2FGC 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 His7 tag used for nickel-affinity chromatography, which was cleaved before reconstitution into liposomes. f, The ATPase activity of E. coli LptB2FGC 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)2FGC 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.