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. Author manuscript; available in PMC: 2023 Dec 11.
Published in final edited form as: Science. 2022 Apr 21;376(6591):383–390. doi: 10.1126/science.abm1183

Fig. 1: Overview of protein machine assembly and rotor component design approaches.

Fig. 1:

(A) (Left) A blueprint of a simple two component machine consisting of an assembly of an axle and a rotor mechanically constrained by the shape of the interface between the two; (Middle) Systematic generation by computational design of a structurally diverse library of machine components and design of interfaces between axle and rotor that mechanically couple the components and direct assembly; (Right) Example of hierarchical design and assembly of a protein machine from axle and rotor components, here a D3 axle and C3 rotor, and interacting interface residues. Wheel-like cyclic DHRs are fused to the end of the axle and rotor components to increase mass, provide a modular handle and a structural signature to monitor conformational variability. (B) Hierarchical design strategies for rotor components (Top) A single chain C1 symmetric and internally C12 symmetric alpha-helical tandem repeat protein is split into three subunits, and each is fused to DHRs via helical fusion (HelixFuse) to generate a C3 rotor (C3_R1) with an internal diameter of 28Å. The 6.0Å cryoEM electron density (shown in grey) shows agreement with the design model (monomer subunits colored by chain); (Middle) A single chain C1 symmetric and internally C24 symmetric alpha-helical tandem repeat protein is split into 4 subunits and each is fused to DHRs to generate a C4 rotor (C4_1) with an internal diameter of 57Å. The 7.9Å cryoEM electron density (shown in grey) shows agreement with the design model (monomer subunits colored by chain); (Bottom) Heterooligomeric helical bundles and DHRs are fused and assembled into a higher-ordered closed C3 structure through helical fusion, after which another round of helical fusion protocol is used to fuse DHRs to each subunit, to generate a C3 rotor (C3_R3) with an internal diameter of 41Å. The negative stain electron density (shown in grey) shows agreement with the design model (monomer subunits colored by chain). Scale bar: 10nm