Fig. 3.

Additional protein density bound to SlpA. (A) Density map of SlpA OMBB trimer at a contour level of 5 σ shown in light blue embedded in a detergent micelle (white). An additional protein density (orange) is partially resolved, present in a subclass of particles seen in 2D class averages. (Scale bars: 50 Å; in Inset, 100 Å.) (B–D) Closeup view of the additional density seen from the Side B, Top C, and Bottom D. The accessory protein adopts a trimeric β-sandwich fold which is aligned with the OMBB without occluding the pore region. (E) Rigid body fit of the AlphaFold2 (37) (AF2) model of the uncharacterized protein DR_0644, which has been previously identified to be associated with SlpA (34), into the extra density not explained by SlpA. The model of the protein fits exceptionally well from the mature N-terminus (N, Gly₂₀) to the C-terminus (C, Arg₂₀₆), with the exception of an unstructured loop (82 to 133), which is in agreement with its low confidence of the pLDDT as measured by AF2. (F and G) The β-sandwich fold of the trimeric DR_0644 (red, orange-red, and orange ribbons) is completed by strand insertion of the first β-strand of the next clockwise oriented subunit, as seen from the Top F and Side G. (H and I) The interface of the SlpA:DR_0644 is stabilized by a prominent salt bridge between SlpA R245 and DR_0644 D186 and further protein:protein interactions such as hydrogen bonding between SlpA N240 and DR_0644 H183. (Scale bars in B–G, 10 Å; in H and I, 5 Å.)