Letter | Published:

Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution

Nature volume 534, pages 724728 (30 June 2016) | Download Citation

Abstract

The interaction of myosin with actin filaments is the central feature of muscle contraction1 and cargo movement along actin filaments of the cytoskeleton2. The energy for these movements is generated during a complex mechanochemical reaction cycle3,4. Crystal structures of myosin in different states have provided important structural insights into the myosin motor cycle when myosin is detached from F-actin5,6,7. The difficulty of obtaining diffracting crystals, however, has prevented structure determination by crystallography of actomyosin complexes. Thus, although structural models exist of F-actin in complex with various myosins8,9,10,11, a high-resolution structure of the F-actin–myosin complex is missing. Here, using electron cryomicroscopy, we present the structure of a human rigor actomyosin complex at an average resolution of 3.9 Å. The structure reveals details of the actomyosin interface, which is mainly stabilized by hydrophobic interactions. The negatively charged amino (N) terminus of actin interacts with a conserved basic motif in loop 2 of myosin, promoting cleft closure in myosin. Surprisingly, the overall structure of myosin is similar to rigor-like myosin structures in the absence of F-actin, indicating that F-actin binding induces only minimal conformational changes in myosin. A comparison with pre-powerstroke and intermediate (Pi-release)7 states of myosin allows us to discuss the general mechanism of myosin binding to F-actin. Our results serve as a strong foundation for the molecular understanding of cytoskeletal diseases, such as autosomal dominant hearing loss and diseases affecting skeletal and cardiac muscles, in particular nemaline myopathy and hypertrophic cardiomyopathy.

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Accessions

Primary accessions

Electron Microscopy Data Bank

Data deposits

The coordinates and electron microscopy density maps have been deposited in the Protein Data Bank (PDB) under accession numbers 5JLF and 5JLH and the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-8162 to EMD-8165.

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Acknowledgements

We thank O. Hofnagel for assistance in cryo sample preparation. We acknowledge R. Matadeen and S. de Carlo for image acquisition at the Netherlands Centre for Nanoscopy in Leiden. We thank R. S. Goody for reading the manuscript. This work was supported by the Max Planck Society, the European Research Council under the European Union’s Seventh Framework Program (FP7/2007-2013) (grant number 615984) (to S.R.), the Behrens-Weise foundation (to S.R.) and German Research Foundation (DFG) grant MA 1081/21-1 (to D.J.M.). J.v.d.E. is a fellow of Studienstiftung des deutschen Volkes.

Author information

Author notes

    • Sarah M. Heissler

    Current address: Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Affiliations

  1. Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany

    • Julian von der Ecken
    •  & Stefan Raunser
  2. Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany

    • Sarah M. Heissler
    • , Salma Pathan-Chhatbar
    •  & Dietmar J. Manstein
  3. Division for Structural Analysis, Hannover Medical School, 30625 Hannover, Germany

    • Dietmar J. Manstein

Authors

  1. Search for Julian von der Ecken in:

  2. Search for Sarah M. Heissler in:

  3. Search for Salma Pathan-Chhatbar in:

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Contributions

D.J.M. and S.R. designed the project. S.M.H. and S.P.-C. purified actin, tropomyosin, and myosin constructs. D.J.M. supervised protein work. J.v.d.E. prepared specimens, recorded, analysed and processed the data, and prepared figures. S.R. managed the project. J.v.d.E. and S.R. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Stefan Raunser.

Reviewer Information Nature thanks E. Nogales, J. Löwe and A. Houdusse for their contribution to the peer review of this work.

Extended data

Supplementary information

Videos

  1. 1.

    Cryo-EM structure of the ATM complex in detail

    a, Cryo-EM reconstruction of F-actin (five central subunits in green and one subunit in cyan) decorated with tropomyosin (blue) and myosin in rigor state (central molecules highlighted in red). Close-up ends with the central part of the map. b, Representative part of the central core region of the F-actin filament shows better than average resolution. c, Interface between D-loop, SD1 of F-actin and HLH motif of myosin. d, Myosin CM-loop bound to SD1 and SD3 of F-actin.

  2. 2.

    Model of the rigor ATM complex and subdomains in F-actin

    F-actin (green, cyan) decorated with tropomyosin (blue) and myosin (red). Subdomain organisation of F-actin is shown.

  3. 3.

    Domain organisation of myosin on F-actin

    Domain organisation of the NM-2C head region (depicted in different colours and labelled as in Fig. 1b-d). Close-ups on HLH motif and surface loops (CM-loop, loop 3, loop 4) at the F-actin-myosin interface.

  4. 4.

    F-actin-myosin interfaces in detail

    a-c, Close-ups on the HLH motif (a), CM-loop (b) and loop 2 (c) of myosin bound to F-actin in rigor state. d, e, A positively charged basin is formed by loop 2, helix-W and the supporting loop of myosin to stabilize the pulled N-terminus conformation of F-actin. Myosin is shown as ribbon with residues (d) or as surface coloured by the electrostatic Coulomb potential ranging from -10 kcal mol-1 in red to +10 kcal mol-1 in blue.

  5. 5.

    Model of weak to strong binding of myosin to F-actin

    a, Myosin binds in a weak PPS state (purple, PDB: 5I4E) to F-actin. b, L50 rotates and binds to F-actin resulting in a stronger bound myosin (Pi-release state in blue). c, d, Finally, U50 rotates and binds to F-actin (c) obtaining the full interface of strongly bound myosin (rigor state in red) on F-actin (d). Models were aligned as shown in Extended Data Fig. 9f. For better visualization, differences in F-actin are not shown and F-actin is depicted only in the M-state (green, cyan).

  6. 6.

    Model of myosin-binding to F-actin in detail

    a, Initial binding of myosin (purple) to F-actin (yellow). b, L50 rotates and binds to F-actin. c, The base of loop 2 (red) is stabilized by hydrophobic interactions with F-actin. d, It also interacts with the negatively charged N-terminus of F-actin inducing its pulling and ordering (A-state in yellow, M-state in cyan). L50 is in its final rigor state conformation. e, Negatively charged strut gets attracted to the positively charged patch of the base of loop 2. U50 rotates and binds to F-actin resulting in the interface observed in the rigor state (red).

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https://doi.org/10.1038/nature18295

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