Wrapped superhelically around actin filaments, the coiled-coil alphahelices of tropomyosin regulate muscle contraction by cooperatively blocking or exposing myosin-binding sites on actin. In non-muscle cells, tropomyosin additionally controls access of actin-binding proteins involved in cytoskeletal actin filament maintenance and remodeling. Tropomyosin's global shape and flexibility play a key role in the assembly, maintenance and regulatory switching of thin filaments, yet remain insufficiently characterized. Here, electron microscopy and molecular dynamics simulations yielded conformations of tropomyosin closely resembling each other. The EM and simulations show that isolated tropomyosin has an average curved conformation with a design well-matched to its superhelical shape on Factin. In addition, they show that tropomyosin bends smoothly yet anisotropically about its distinctive helically curved conformation, without any signs of unfolding, chain separation, localized kinks or joints. Previous measurements, assuming tropomyosin to be straight on average, mistakenly suggested considerable flexibility (with persistence lengths only ~3 times the protein's length). However, taking the curved average structure determined here as reference for the flexibility measurements yields a persistence length of ~25 lengths, revealing that tropomyosin actually is semi-rigid. Corresponding simulation of a triple mutant (A74L-A78V-A81L) with weak actin-affinity shows it lacks shape-complementarity to F-actin. Thus tropomyosin's pre-shaped, semi-rigid architecture is essential for the assembly of actin-filaments. Further, we propose that once bound to thin filaments, tropomyosin will be stiff enough to act as a cooperative unit and move on actin in a concerted way between known regulatory states.
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