(A) Cryo-EM map of Δ30-Msp1E214Q showing the arrangement of the fishhook motifs in the spiral. M1-M5 shows significant density for the entire fishhook motif (α0 and the L1), whereas M6 shows density for L1 but not for α0. On the left, the structure is displayed at σ = 5.5, showing the fishhook motifs of different subunits radially organized with their N-termini pointing to the center of the spiral. On the right, the structure is displayed at σ = 3.8, showing the density of the central hub (cyan) emerge where the α0s of M1-M5 converge in a staggered alignment. (B and C) The electrostatic potential surface of the Δ30-Msp1E214Q structure shows that Msp1 displays a positively charged surface. Positive charges are colored in blue, negative charges in red, and neutral side chains in white. (D) Surface representation of individual subunits highlighting amino acids in the LD likely to engage the hydrophobic substrate. These amino acids are buried in by α0 in M2-M5 but exposed in M6 where α0 is melted. The labeled amino acids include the previously identified L89, Y92, E93, V101, P103, I106, D112, I113, G114, G115, I116, and other hydrophobic amino acids L87, V88, V96, A97, L98, V100, A102, P107, V108, F110. (E) Mapping of amino acids that interact with the substrate (identified in Li et al. (2019) by crosslinking or immunoprecipitation) to the Δ30-Msp1E214Q structure shows that on M6, they form a patch at the seam of the spiral. The central hub is colored in cyan, α0 in green, L1 in blue, previously identified amino acids that interact with the substrate in gold, and other hydrophobic amino acids in the LD in magenta.