Swinging Lever Mechanism of Myosin Directly Shown by Time-Resolved Cryo-EM

Authors

David P. Klebl, University of Leeds
Sean N. McMillan, University of Leeds
Cristina Risi, Macon & Joan Brock Virginia Health Sciences at Old Dominion UniversityFollow
Eva Forgacs, Macon & Joan Brock Virginia Health Sciences at Old Dominion UniversityFollow
Betty Virok, Macon & Joan Brock Virginia Health Sciences at Old Dominion University
Jennifer L. Atherton, Macon & Joan Brock Virginia Health Sciences at Old Dominion UniversityFollow
Sarah A. Harris, University of Sheffield
Michele Stofella, University of Leeds
Donald A. Winkelmann, Rutgers University - New Brunswick/Piscataway
Frank Sobott, University of Leeds
Vitold E. Galkin, Macon & Joan Brock Virginia Health Sciences at Old Dominion UniversityFollow
Peter J. Knight, University of Leeds
Stephen P. Muench, University of Leeds
Charlotte A. Scarfi, University of Leeds
Howard D. White, Macon & Joan Brock Virginia Health Sciences at Old Dominion UniversityFollow

ORCID

0000-0002-3145-2072 (Risi), 0009-0005-3283-0492 (Forgacs), 0000-0003-1138-7565 (Galkin),

Document Type

Article

Publication Date

2025

DOI

10.1038/s41586-025-08876-5

Publication Title

Nature

Volume

Article in Press

Pages

24 pp.

Abstract

Myosins produce force and movement in cells through interactions with F-actin¹. Generation of movement is thought to arise through actin-catalysed conversion of myosin from an ATP-generated primed (pre-powerstroke) state to a post-powerstroke state, accompanied by myosin lever swing²,³. However, the initial, primed actomyosin state has never been observed, and the mechanism by which actin catalyses myosin ATPase activity is unclear. Here, to address these issues, we performed time-resolved cryogenic electron microscopy (cryo-EM)⁴ of a myosin-5 mutant having slow hydrolysis product release⁵,⁶. Primed actomyosin was predominantly captured 10 ms after mixing primed myosin with F-actin, whereas post-powerstroke actomyosin predominated at 120 ms, with no abundant intermediate states detected. For detailed interpretation, cryo-EM maps were fitted with pseudo-atomic models. Small but critical changes accompany the primed motor binding to actin through its lower 50-kDa subdomain, with the actin-binding cleft open and phosphate release prohibited. Amino-terminal actin interactions with myosin promote rotation of the upper 50-kDa subdomain, closing the actin-binding cleft, and enabling phosphate release. The formation of interactions between the upper 50-kDa subdomain and actin creates the strong-binding interface needed for effective force production. The myosin-5 lever swings through 93°, predominantly along the actin axis, with little twisting. The magnitude of lever swing matches the typical step length of myosin-5 along actin⁷. These time-resolved structures demonstrate the swinging lever mechanism, elucidate structural transitions of the power stroke, and resolve decades of conjecture on how myosins generate movement.

Rights

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Data Availability

Article states: "The electron density maps and atomic models for unbound primed myosin-5, primed actomyosin-5 and postPS actomyosin-5 have been deposited into the Electron Microscopy Data Bank (EMDB), with the accession codes EMD-19031, EMD-19013 and EMD-19030, and the PDB with the accession codes 8RBG, 8R9V and 8RBF, respectively. The electron density map for rigor actomyosin-5 has been deposited into the EMDB with the accession code EMD-50594. The following models were used for comparison purposes in our study—actomyosin-5 rigor structures PDB IDs: 7PLT, 7PLU, 7PLV, 7PLW and 7PLZ; and actomyosin-5 strong-ADP structures PDB IDs: 7PM5, 7PM6, 7PM7, 7PM8 and 7PM9. Each MD trajectory and parameter–topology file for primed and postPS structures for each MD simulation replicate is provided along with the starting models at figshare via https://doi.org/10.6084/m9.figshare.24948180 (ref. 59)."

Original Publication Citation

Klebl, D. P., McMillan, S. N., Risi, C., Forgacs, E., Virok, B., Atherton, J. L., Harris, S. A., Stofella, M., Winkelmann, D. A., Sobott, F., Galkin, V. E., Knight, P. J., Muench, S. P., Scarff, C. A., & White, H. D. (2025). Swinging lever mechanism of myosin directly shown by time-resolved cryo-EM. Nature. Advance online publication. https://doi.org/10.1038/s41586-025-08876-5

Repository Citation

Klebl, D. P., McMillan, S. N., Risi, C., Forgacs, E., Virok, B., Atherton, J. L., Harris, S. A., Stofella, M., Winkelmann, D. A., Sobott, F., Galkin, V. E., Knight, P. J., Muench, S. P., Scarff, C. A., & White, H. D. (2025). Swinging lever mechanism of myosin directly shown by time-resolved cryo-EM. Nature. Advance online publication. https://doi.org/10.1038/s41586-025-08876-5