Fig. 6: Conformational dynamics of EsxE-EsxF pore assembly revealed by electron microscopy.

a Pentameric 3D reconstruction from negatively stained EsxEF particles. The EsxEF homology model was fit to the density using rigid-body fitting in Chimera. Shown is the bottom view. The electron density map for EsxEF is deposited in the electron microscopy data bank (EMDB code: EMD-22727). b Side view of the 3D reconstruction shown in a. c Putative oligomeric interface formed by the WXG motif of EsxF (green) and the CTD of EsxE (orange). d Differential scanning fluorimetry of the indicating proteins, indicating the thermostability of the proteins and the water accessibility of the tryptophan residues. e Reference-free 2D class averages of the indicated EsxE-EsxF protein particles obtained by negative stain electron microscopy ranked by solvation as determined in e. The complete class averages are shown in Fig. S7. Data are from a single EM session. Representative images of at least two experiments are shown. f Contour map of the solvation value (350 nm/330 nm) obtained from d versus the total number of channel insertions (Fig. S6) for each examined EsxE-EsxF variant. The thresholds were determined based on the event frequency. The WXG variants of EsxE-EsxF form immature pores whereas “mature channel” were formed by wt EsxE-EsxF. The total number of insertions for each protein at each conductance value was smoothed and normalized in Graphpad. Smoothed data were then plotted against the respective solvation values using SigmaPlot. Source data are provided in the Source data file.