Fig. 6. Low-resolution BAM/MBP-EspP structure from in vitro refolding assay.

a Summary of the in vitro assay used for inserting MBP-EspP into BAM-nanodiscs. This figure is an adaptation from Imai et al.⁴⁸ and is used under a CC BY 4.0 license; the image was modified to reflect the use of MBP-EspP in this set of experiments. b SEC trace of Bam/MBP-EspP purification after in vitro refolding. The green allow indicates the elution of the complex, while the purple arrow indicates the elution of excess surA. The inset shows SDS-PAGE analysis demonstrating the purity of the MBP-EspP inclusion bodies used for refolding. Source data are provided as a Source Data file. c SDS-PAGE gel of the MBP-EspP/BAM-inserted nanodiscs used for EM studies. The black triangle indicates the MSP protein while the blue triangle indicates MBP-EspP. Source data are provided as a Source Data file. d Negative-stain image of the MBP-EspP/BAM-inserted nanodiscs. The scale bar represents 50 nm. e A cutaway view of the 7.0 Å resolution BAM/MBP-EspP structure showing enlarged nanodisc density and triangular shape of the hybrid-barrel compared to the BAM-only structure. The position of the BamA barrel in the BAM-only structure is indicated by the black dashed oval; the position of the nanodisc density for the BAM-only structure is indicated by the gray oval. f The map from the cryoEM reconstruction to 7 Å resolution, with each component color coded. g The segmented map with the nanodisc density removed for clarity, depicting the extra density for the MBP-EspP substrate (gray map with ribbon in purple outline). h The nanodisc BAM/MBP-EspP intermediate complex highlighting only the density for the MBP-EspP component, modeled as four strands of the EspP barrel with the N-terminal region trailing down under BAM to a globular region of density. i A simplified view from under the hybrid-barrel. j Zoomed view of the triangular shape of BamA/EspP hybrid-barrel. k Zoomed view of the modeled four strands of EspP integrated into the barrel of BamA.