Compatibility of our model with previous structures. (A) Comparison of the prepore and pore rims. Domains 1a and 3 from the prepore (cyan/orange) and pore (blue/red) are overlaid. The RMSDon Cαs for the top of the rim (seven domains 1a, residues 176–250) and the rim (seven domains 1a and 3, residues 176–250 + residues 458–496) is 1.48 Å. The black ring denotes the outer limit of LF contacts, and the blue “N”s denoted the innermost contacts. (B) Hypothetical model of an assembly intermediate, a PA₈₃–PA₆₃–LF complex. Starting with a (PA₆₃)₂–LF moiety extracted from our EM structure, the location of PA₂₀ was predicted by overlaying the crystal structure of domain 1 of full-length PA₈₃ onto domain 1a of the counterclockwise PA₆₃ protomer, creating a new PA₈₃ protomer (shown in orange and red; as described as in Material and methods section Modeling the (PA₆₃)₇–(LF)₃ complex). No major steric clashes were observed between the new PA₂₀ moiety and the clockwise PA₆₃ protomer (green), which makes the major interactions with LF. However, only the closed conformer of LF is compatible with the presence of PA₂₀. The circled “N” marks the first ordered helix of closed LF, which is sterically occluded from reorganizing and entering the channel pore. The yellow dashed line indicates novel interfaces that could stabilize the intermediate.