Cryo-EM structure of mastigoneme. (A) Cartoon diagram illustrating the domain organization of MST1. The N-terminal consists of nine Ig-like fold repeats (Ig 1–9), followed by the Ig to Sushi linker region (IS linker), six SDs (SDa–f), the PPII Helix, and the C-terminal Helix. (B) Overall cryo-EM map and ribbon representation of the MST1 protein. The colors correspond to the domains shown in the cartoon in A. (C) The fundamental symmetry operator involves dimeric pairing, spanning ∼38.2 nm in length. (D) Six adjacent MST1 molecules (labeled from A to F) are positioned with an ∼76.8-nm shift along the filament axis. The filament displays non-polarity, and in either direction, a mastigoneme dimer ascends ∼19.2 nm with a 90° twist, repeating its pattern. This configuration encompasses dimeric pairing interfaces (grey cycle), supportive pairing interfaces (yellow cycle), and anti-parallel pairing interfaces (green cycle). Along the filament axis, the mastigoneme reveals varying thickness regions, with some measuring around 6 nm in diameter and others ∼10 nm in diameter. These distinctive features are also discernible in the cryo-EM 2D images.