Fig. 2: Design of axle machine components.

(A) Hierarchical design of a D3 symmetric homohexamer axle (D3_3). Parametric design of interdigitated helices in D3 symmetry is achieved by sampling supercoil radius (R₁,R₂), helical phase (Δφ_1-1, Δφ_1-2), supercoil phase (Δφ_0-1,Δφ_0-2) of two helical fragments, and the z-offset (Z_off and supercoil twist (ω₀). The interface is designed using the HBNet protocol to identify hydrogen-bond networks spanning the 6 helices mediating high-order specificity. The design is then fused to C3 wheel-like homotrimers using RosettaRemodel. The 4.2Å cryoEM electron density is consistent with the design model (B) Hierarchical design of a D8 axle (D8_1). Interdigitated helical extensions at the termini of a parametrically designed C8 homohexamer are sampled using Rosetta BluePrintBuilder and hydrogen bond networks are identified using HBnet, while sampling rotation and translation in D8 symmetry using Rosetta SymDofMover. The 7.4Å cryoEM electron density is in close agreement with the design model; (C) Hierarchical design of a C3 homotrimer axle (C3_A1). A parametrically designed C3 homotrimer was circularly permutated and an extra heptad repeat added to increase the aspect ratio, after DHRs were fused to each subunit using Hfuse. The negative stain electron density is consistent with the design model (D) Additional axle components overlaid with experimental negative stain electron density, corresponding to D2 (D2_2), D4 (D4_2), D5 (D5_2), C8 (C8_1) and D8 (D8_3) designs. Model monomer subunits are colored by chain, and electron densities are shown as grey surfaces. Scale bar: 10 nm