Experiments to validate that the ESX-3 core complex extracted from actively secreting mycobacteria is the same as the complex for which the structure was solved. We investigated the expression of the ESX-3 complex in the mycobacterial membrane, the stoichiometry of the extracted and purified complex and the orientation of the complex in the membrane. a, Overview of the constructs used in secretion assays and for the validation of the structure of the ESX-3 core complex. Plasmid pMyNT:ESX-3 encodes the ESX-3 gene cluster under acetamidase promoter control, and pMyNT:ESX-3i under control of the native IdeR promotor. The expression of the latter construct is induced in iron-depleted culture medium. b, BN–PAGE and western blot analyses (EccC3-His) indicate the presence of the 900-kDa ESX-3 core complex in the membrane of secretion-competent cells that contain the ESX-3 gene cluster under control of either the acetamidase promoter or the native IdeR promoter (see a). The gel represents two independent experiments. c, Comparison of size-exclusion chromatography profiles of ESX-3 core complexes purified from secretion-competent cells transformed with plasmids pMyNT:ESX-3 and pMyNT:ESX-3i (both expressed in M. smegmatis ΔESX-3; see a), with the ESX-3 core complex purified from the minimal expression construct (pMyNT:Mini) expressed in wild-type M. smegmatis, which was used to determine the cryo EM structure (Extended Data Fig. 1a). The chromatograms are representative of three independent purifications. d, ESX-3 core complexes purified from secretion-competent M. smegmatis ΔESX-3 transformed with either pMyNT:ESX-3 or pMyNT:ESX-3i, in the presence and absence of β-mercaptoethanol. A disulfide bridge is found in the EccB3 protein in complexes purified from secretion-competent cells. The two positions for EccB3 in SDS–PAGE are indicated. In the structure of the ESX-3 core complex, EccB3 is periplasmic and contributes to the assembly between protomers. We observe a different mobility for EccB3 in the ESX-3 core complex purified from secretion-competent cells when the SDS–PAGE is performed in the presence or absence of β-mercaptoethanol. This is compatible with an intramolecular disulfide bridge in EccB3, as expected in the oxidative environment of the periplasm. The gel represents two independent experiments. e, Determination of the stoichiometry of ESX-3 complexes in secretion-competent M. smegmatis ΔESX-3 transformed with either pMyNT:ESX-3 or pMyNT:ESX-3i. Oriole staining of the SDS–PAGE bands—in order to estimate the ratio of subunits obtained—showed that EccD3 is over-represented, in agreement with the observed 1:1:2:1 stoichiometry in the structure of the ESX-3 core complex. SDS–PAGE shows that the density of the bands of EccD3 is higher than those of EccE3, despite both proteins having a similar molecular mass. The intensities of the bands were integrated and the relative ratio of subunits was estimated, taking into account the differences in molecular mass of the proteins. In the bar chart, the bars are the reported means and the standard deviations are shown as dots. The results indicated a stoichiometry of 1:1:2:1 (EccB3:EccC3:EccD3:EccE3). The gel shows three technical replicates and is representative of two independent experiments. f, Analysis of the composition of the protein complex subunit using iBAQ. The size of the dots correlates with the number of identified unique peptides. The iBAQ–MS method was, in our experiments, not sufficiently sensitive to define the stoichiometry of ESX-3. We used the ESX-3 complex from our structural studies as an internal control because the stoichiometry of the ESX-3 structure was fully determined as 1:1:2:1 at the high resolution of our cryo-EM maps. The iBAQ method estimates a 1:1:1:1 stoichiometry for the purified complex that we use to resolve the structure by cryo-EM, thus indicating that the method does not report the correct stoichiometry for ESX-3 in these experiments. When the stoichiometry of the native ESX-3 core complex in secretion-competent cells was estimated by the iBAQ method used previously for the ESX-5 core complex19, a 1:1:1:1 ratio was also obtained (data not shown). g, Analysis of purified EccD3 using size-exclusion chromatography (Superose-6 Increase 10/300 GL column) coupled with multi-angle light scattering. The lines show the deconvolution of the contributions of the EccD3–DDM protein–detergent complex (dot-dashed line), the protein (dashed line) and the detergent micelles (dotted line) to the total mass of the complex. This experiment demonstrates that EccD3 dimerizes as observed in structure of the ESX-3 core complex. The experiment is representative of two technical replicates. h, Analysis of the derivatives of the ESX-3 core complex by size-exclusion chromatography. Removing the periplasmic fork of EccB3 or EccE3 leads to dissociation towards single protomers, showing that both components are essential for the stability of the ESX-3 complex. The elution peak of the ESX-3 core complex dimer is indicated. The chromatograms are representative of two independent purifications. i, Isolated mycobacterial membranes from wild-type M. smegmatis containing the ‘high yield’ minimal expression construct (pMyNT:Mini) and extraction of ESX-3 core complexes after crosslinking using DSS. ESX-3 complexes were analysed by BN–PAGE and western blot analyses (EccD3–StrepII). As a control, complexes formed by EccD3 expressed alone were also analysed simultaneously. Only ESX-3 assembled into large complexes, including those of higher molecular mass (higher MW species) than the ESX-3 dimer. The gel is representative of two independent experiments.