a–e, T6SS operon genomic organization and constructs used for in vitro analyses. a, Schematic representation of the T6SS sci-1 gene cluster from entero aggregative E. coli. The numbers on top refer to the gene locus tag (EC042_XXXX). Genes encoding core components (identified by their names on bottom, for example, ‘B’ refers to the tssB gene) are coloured grey. Genes of unknown function are coloured white. The three genes used to reconstitute the core membrane complex are coloured orange (tssJ), blue (tssL) and green (tssM). b, Schematic representation of the engineered constructs: the tssJ, tssL and tssM genes were amplified with an additional Shine Dalgarno (SD) sequence and 3′ StrepII, 5′ Flag and 5′ 6×His tags respectively. These three fragments were cloned into the pRSF-Duet vector (c). This construct allows the production of the C-terminally StrepII-tagged TssJ outer membrane (OM) lipoprotein and N-terminally Flag-tagged TssL and 6×His-tagged TssM inner-membrane (IM) proteins (d, e). The proteins are schematized and their boundaries and principal characteristics (TM, transmembrane segments; SP, signal peptide; CYS, acylated cysteine) are indicated (d) and their topologies are shown (e). The additional TssM constructs (TssMp, TssM32Ct and TssM26Ct) used for SAXS or X-ray analyses are shown at the bottom. f–h, Purification and biochemical characterization of the T6SS membrane core complex. f, Analytical size-exclusion chromatography analysis of the purified TssJLM complex (continuous line) on a Superose 6 column, calibrated with 75-, 158-, 440- and 660-kDa molecular mass markers (dotted lines). The molecular mass of each marker (in kilodaltons) is indicated on the top of the corresponding peak. An arrow indicates the position of the peak fraction corresponding to the TssJLM complex. g, SDS–PAGE of the purified TssJLM complex analysed by Coomassie staining (CB) or immunoblotting using anti-His (α-His), -Flag (α-Flag) and -StrepII (α-STREP) antibodies. h, Left: cysteine labelling of the purified TssJLM complex in reducing and denaturing conditions as described in Methods. The total number of cysteine residues was nine for TssM, five for TssL and none for TssJ (the N-terminal cysteine is acylated). Right: the relative amount of TssL compared with TssM (densitometry relative to the number of free cysteine residues, fixed at 1 for TssM).