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Cryo-EM structure of the human cardiac myosin filament

Nature volume 623, pages 853–862 (2023)Cite this article

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Abstract

Pumping of the heart is powered by filaments of the motor protein myosin that pull on actin filaments to generate cardiac contraction. In addition to myosin, the filaments contain cardiac myosin-binding protein C (cMyBP-C), which modulates contractility in response to physiological stimuli, and titin, which functions as a scaffold for filament assembly1. Myosin, cMyBP-C and titin are all subject to mutation, which can lead to heart failure. Despite the central importance of cardiac myosin filaments to life, their molecular structure has remained a mystery for 60 years2. Here we solve the structure of the main (cMyBP-C-containing) region of the human cardiac filament using cryo-electron microscopy. The reconstruction reveals the architecture of titin and cMyBP-C and shows how myosin’s motor domains (heads) form three different types of motif (providing functional flexibility), which interact with each other and with titin and cMyBP-C to dictate filament architecture and function. The packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps to generate the cardiac super-relaxed state3; how titin and cMyBP-C may contribute to length-dependent activation4; and how mutations in myosin and cMyBP-C might disturb interactions, causing disease5,6. The reconstruction resolves past uncertainties and integrates previous data on cardiac muscle structure and function. It provides a new paradigm for interpreting structural, physiological and clinical observations, and for the design of potential therapeutic drugs.

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Fig. 1: Single-particle 3D reconstruction of the C-zone reveals the organization of titin, cMyBP-C and myosin heads and tails.
Fig. 2: Model building and fitting explains all of the observed densities in the cryo-EM map.
Fig. 3: Myosin tails form an interconnected network in the filament backbone.
Fig. 4: Titin functions as a template for myosin organization.
Fig. 5: cMyBP-C interacts with myosin tails and heads.
Fig. 6: IHM interactions may stabilize the relaxed state.

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Data availability

Structural data supporting the findings of this study on the structure of the human cardiac thick filament have been deposited at the EMDB. One two-crown and two five-crown maps have been deposited under accession codes EMD-29734, EMD-29722 and EMD-29726, respectively. An atomic model of the full three-crown structure, with myosin heads, tails, titins and cMyBP-C, has been deposited at the PDB under accession code 8G4L. PDB data used to build the model were 5N69 and 2FXO. AlphaFold structures used to build the model were P10916, P12883 and Q14896.

References

  1. Wang, L., Geist, J., Grogan, A., Hu, L. R. & Kontrogianni-Konstantopoulos, A. Thick filament protein network, functions, and disease association. Compr. Physiol. 8, 631–709 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Huxley, H. E. Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. J. Mol. Biol. 7, 281–308 (1963).

    Article  CAS  PubMed  Google Scholar 

  3. Hooijman, P., Stewart, M. A. & Cooke, R. A new state of cardiac myosin with very slow ATP turnover: a potential cardioprotective mechanism in the heart. Biophys. J. 100, 1969–1976 (2011).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. de Tombe, P. P. et al. Myofilament length dependent activation. J. Mol. Cell. Cardiol. 48, 851–858 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Alamo, L. et al. Effects of myosin variants on interacting-heads motif explain distinct hypertrophic and dilated cardiomyopathy phenotypes. eLife https://doi.org/10.7554/eLife.24634 (2017).

  6. Nag, S. et al. The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations. Nat. Struct. Mol. Biol. 24, 525–533 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Geeves, M. A. & Holmes, K. C. Structural mechanism of muscle contraction. Annu. Rev. Biochem. 68, 687–728 (1999).

    Article  CAS  PubMed  Google Scholar 

  8. Kensler, R. W. The mammalian cardiac muscle thick filament: crossbridge arrangement. J. Struct. Biol. 149, 303–312 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Craig, R. & Woodhead, J. L. Structure and function of myosin filaments. Curr. Opin. Struct. Biol. 16, 204–212 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Wendt, T., Taylor, D., Trybus, K. M. & Taylor, K. Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2. Proc. Natl Acad. Sci. USA 98, 4361–4366 (2001).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Woodhead, J. L. et al. Atomic model of a myosin filament in the relaxed state. Nature 436, 1195–1199 (2005).

    Article  ADS  CAS  PubMed  Google Scholar 

  12. Alamo, L. et al. Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity. J. Mol. Biol. 384, 780–797 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lee, K. H. et al. Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals. Proc. Natl Acad. Sci. USA 115, E1991–E2000 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Nelson, S., Beck-Previs, S., Sadayappan, S., Tong, C. & Warshaw, D. M. Myosin-binding protein C stabilizes, but is not the sole determinant of SRX myosin in cardiac muscle. J. Gen. Physiol. https://doi.org/10.1085/jgp.202213276 (2023).

  15. Cooke, R. The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle. Biophys. Rev. 3, 33–45 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Anderson, R. L. et al. Deciphering the super relaxed state of human beta-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers. Proc. Natl Acad. Sci. USA 115, E8143–E8152 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zoghbi, M. E., Woodhead, J. L., Moss, R. L. & Craig, R. Three-dimensional structure of vertebrate cardiac muscle myosin filaments. Proc. Natl Acad. Sci. USA 105, 2386–2390 (2008).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Al-Khayat, H. A., Kensler, R. W., Squire, J. M., Marston, S. B. & Morris, E. P. Atomic model of the human cardiac muscle myosin filament. Proc. Natl Acad. Sci. USA 110, 318–323 (2013).

    Article  ADS  CAS  PubMed  Google Scholar 

  19. Tamborrini, D. et al. Structure of the native myosin filament in the relaxed cardiac sarcomere. Nature https://doi.org/10.1038/s41586-023-06690-5 (2023).

  20. Padron, R., Dutta, D. & Craig, R. Variants of the myosin interacting-heads motif. J. Gen. Physiol. https://doi.org/10.1085/jgp.202213249 (2023).

  21. Hu, Z., Taylor, D. W., Reedy, M. K., Edwards, R. J. & Taylor, K. A. Structure of myosin filaments from relaxed Lethocerus flight muscle by cryo-EM at 6 A resolution. Sci. Adv. 2, e1600058 (2016).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  22. Koubassova, N. A. et al. Interacting-heads motif explains the X-ray diffraction pattern of relaxed vertebrate skeletal muscle. Biophys. J. 121, 1354–1366 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  23. McLachlan, A. D. & Karn, J. Periodic charge distributions in the myosin rod amino acid sequence match cross-bridge spacings in muscle. Nature 299, 226–231 (1982).

    Article  ADS  CAS  PubMed  Google Scholar 

  24. Taylor, K. C. et al. Skip residues modulate the structural properties of the myosin rod and guide thick filament assembly. Proc. Natl Acad. Sci. USA 112, E3806–E3815 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wray, J. S. Structure of the backbone in myosin filaments of muscle. Nature 277, 37–40 (1979).

    Article  ADS  CAS  PubMed  Google Scholar 

  26. Squire, J. M. General model of myosin filament structure. 3. Molecular packing arrangements in myosin filaments. J. Mol. Biol. 77, 291–323 (1973).

    Article  CAS  PubMed  Google Scholar 

  27. Gregorio, C. C., Granzier, H., Sorimachi, H. & Labeit, S. Muscle assembly: a titanic achievement? Curr. Opin. Cell Biol. 11, 18–25 (1999).

    Article  CAS  PubMed  Google Scholar 

  28. Tskhovrebova, L. et al. Shape and flexibility in the titin 11-domain super-repeat. J. Mol. Biol. 397, 1092–1105 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. Harris, S. P., Lyons, R. G. & Bezold, K. L. In the thick of it: HCM-causing mutations in myosin binding proteins of the thick filament. Circ. Res. 108, 751–764 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Flashman, E., Redwood, C., Moolman-Smook, J. & Watkins, H. Cardiac myosin binding protein C: its role in physiology and disease. Circ. Res. 94, 1279–1289 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Granzier, H. L. & Labeit, S. The giant protein titin: a major player in myocardial mechanics, signaling, and disease. Circ. Res. 94, 284–295 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Bucher, R. M., Svergun, D. I., Muhle-Goll, C. & Mayans, O. The structure of the FnIII Tandem A77-A78 points to a periodically conserved architecture in the myosin-binding region of titin. J. Mol. Biol. 401, 843–853 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Lee, K., Harris, S. P., Sadayappan, S. & Craig, R. Orientation of myosin binding protein C in the cardiac muscle sarcomere determined by domain-specific immuno-EM. J. Mol. Biol. 427, 274–286 (2015).

    Article  CAS  PubMed  Google Scholar 

  34. Tonino, P., Kiss, B., Gohlke, J., Smith III, J. E. & Granzier, H. Fine mapping titin’s C-zone: matching cardiac myosin-binding protein C stripes with titin’s super-repeats. J. Mol. Cell. Cardiol. 133, 47–56 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Huxley, H. E. & Brown, W. The low-angle x-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor. J. Mol. Biol. 30, 383–434 (1967).

    Article  CAS  PubMed  Google Scholar 

  36. Sohn, R. L. et al. A 29 residue region of the sarcomeric myosin rod is necessary for filament formation. J. Mol. Biol. 266, 317–330 (1997).

    Article  CAS  PubMed  Google Scholar 

  37. Houmeida, A., Holt, J., Tskhovrebova, L. & Trinick, J. Studies of the interaction between titin and myosin. J. Cell Biol. 131, 1471–1481 (1995).

    Article  CAS  PubMed  Google Scholar 

  38. Muhle-Goll, C. et al. Structural and functional studies of titin’s fn3 modules reveal conserved surface patterns and binding to myosin S1—a possible role in the Frank-Starling mechanism of the heart. J. Mol. Biol. 313, 431–447 (2001).

    Article  CAS  PubMed  Google Scholar 

  39. McNamara, J. W. et al. Ablation of cardiac myosin binding protein-C disrupts the super-relaxed state of myosin in murine cardiomyocytes. J. Mol. Cell. Cardiol. 94, 65–71 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Heling, L., Geeves, M. A. & Kad, N. M. MyBP-C: one protein to govern them all. J. Muscle Res. Cell Motil. 41, 91–101 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Luther, P. K. et al. Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle. Proc. Natl Acad. Sci. USA 108, 11423–11428 (2011).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  42. Reconditi, M. et al. Sarcomere-length dependence of myosin filament structure in skeletal muscle fibres of the frog. J. Physiol. 592, 1119–1137 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Freiburg, A. & Gautel, M. A molecular map of the interactions between titin and myosin-binding protein C. Implications for sarcomeric assembly in familial hypertrophic cardiomyopathy. Eur. J. Biochem. 235, 317–323 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Bennett, P., Rees, M. & Gautel, M. The axial alignment of titin on the muscle thick filament supports its role as a molecular ruler. J. Mol. Biol. 432, 4815–4829 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Craig, R. & Padron, R. Structural basis of the super- and hyper-relaxed states of myosin II. J. Gen. Physiol. https://doi.org/10.1085/jgp.202113012 (2022).

  46. Grinzato, A. et al. Cryo-EM structure of the folded-back state of human β-cardiac myosin. Nat. Commun. 14, 3166 (2023).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lowey, S., Saraswat, L. D., Liu, H., Volkmann, N. & Hanein, D. Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins. J. Mol. Biol. 371, 902–913 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Blankenfeldt, W., Thoma, N. H., Wray, J. S., Gautel, M. & Schlichting, I. Crystal structures of human cardiac β-myosin II S2-Δ provide insight into the functional role of the S2 subfragment. Proc. Natl Acad. Sci. USA 103, 17713–17717 (2006).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ait-Mou, Y. et al. Titin strain contributes to the Frank-Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins. Proc. Natl Acad. Sci. USA 113, 2306–2311 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  50. Brunello, E. et al. Myosin filament-based regulation of the dynamics of contraction in heart muscle. Proc. Natl Acad. Sci. USA 117, 8177–8186 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  51. Alamo, L. et al. Conserved intramolecular interactions maintain myosin interacting-heads motifs explaining tarantula muscle super-relaxed state structural basis. J. Mol. Biol. 428, 1142–1164 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Ma, W. et al. The super-relaxed state and length dependent activation in porcine myocardium. Circ. Res. 129, 617–630 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Reconditi, M. et al. Myosin filament activation in the heart is tuned to the mechanical task. Proc. Natl Acad. Sci. USA 114, 3240–3245 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  54. Irving, M. Regulation of contraction by the thick filaments in skeletal muscle. Biophys. J. 113, 2579–2594 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  55. Espinoza-Fonseca, L. M., Kast, D. & Thomas, D. D. Molecular dynamics simulations reveal a disorder-to-order transition on phosphorylation of smooth muscle myosin. Biophys. J. 93, 2083–2090 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Blair, C. A. et al. A Protocol for collecting human cardiac tissue for research. VAD J. https://doi.org/10.13023/VAD.2016.12 (2016).

  57. Chaponnier, C., Janmey, P. A. & Yin, H. L. The actin filament-severing domain of plasma gelsolin. J. Cell Biol. 103, 1473–1481 (1986).

    Article  CAS  PubMed  Google Scholar 

  58. Hidalgo, C., Padron, R., Horowitz, R., Zhao, F. Q. & Craig, R. Purification of native myosin filaments from muscle. Biophys. J. 81, 2817–2826 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Craig, R. Isolation, electron microscopy and 3D reconstruction of invertebrate muscle myofilaments. Methods 56, 33–43 (2012).

    Article  CAS  PubMed  Google Scholar 

  60. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  61. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  62. Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun. Biol. 4, 874 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Kidmose, R. T. et al. Namdinator—automatic molecular dynamics flexible fitting of structural models into cryo-EM and crystallography experimental maps. IUCrJ 6, 526–531 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  65. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  66. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019).

    Article  ADS  CAS  Google Scholar 

  67. Lopez-Blanco, J. R. & Chacon, P. iMODFIT: efficient and robust flexible fitting based on vibrational analysis in internal coordinates. J. Struct. Biol. 184, 261–270 (2013).

    Article  PubMed  Google Scholar 

  68. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D 74, 519–530 (2018).

    Article  ADS  CAS  Google Scholar 

  69. Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    Article  CAS  PubMed  Google Scholar 

  70. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  71. Maw, M. C. & Rowe, A. J. Fraying of A-filaments into three subfilaments. Nature 286, 412–414 (1980).

    Article  ADS  CAS  PubMed  Google Scholar 

  72. Squire, J. M. & Knupp, C. X-ray diffraction studies of muscle and the crossbridge cycle. Adv. Protein Chem. 71, 195–255 (2005).

    Article  CAS  PubMed  Google Scholar 

  73. Belus, A. et al. The familial hypertrophic cardiomyopathy-associated myosin mutation R403Q accelerates tension generation and relaxation of human cardiac myofibrils. J. Physiol. 586, 3639–3644 (2008).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  74. Takezawa, Y. et al. Backward movements of cross-bridges by application of stretch and by binding of MgADP to skeletal muscle fibers in the rigor state as studied by X-ray diffraction. Biophys. J. 76, 1770–1783 (1999).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  75. Woodhead, J. L. & Craig, R. Through thick and thin–interfilament communication in muscle. Biophys. J. 109, 665–667 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  76. Brito, R. et al. A molecular model of phosphorylation-based activation and potentiation of tarantula muscle thick filaments. J. Mol. Biol. 414, 44–61 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank C. Ouch, K. Song and C. Xu for help and training in cryo-EM imaging; K. H. Lee and G. Hendricks for conventional EM training; N. Grigorieff for use of the Leica EM GP2 plunge freezer; T. Irving and W. Ma for the unpublished X-ray diffraction pattern of porcine cardiac muscle shown in Extended Data Fig. 3; and C. Cremo for the gift of the gelsolin N-terminal-half plasmid. This work was supported by NIH grants AR072036, HL139883, HL164560, AR081941, HL149164 and HL148785. UCSF ChimeraX, used for molecular graphics, was developed by the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco, with support from NIH grant GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.

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Authors and Affiliations

  1. Division of Cell Biology and Imaging, Department of Radiology, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Debabrata Dutta, Vu Nguyen, Raúl Padrón & Roger Craig

  2. Department of Physiology and Division of Cardiovascular Medicine, University of Kentucky, Lexington, KY, USA

    Kenneth S. Campbell

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  1. Debabrata Dutta
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  2. Vu Nguyen
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Contributions

D.D. prepared samples, performed cryo-EM and carried out reconstruction, atomic fitting, refinement, structure analysis and database deposition. V.N. provided computational expertise and analysed data. K.S.C. provided curated human heart tissue. R.C. and R.P. performed analysis of the structure, co-wrote the paper and obtained funding.

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Correspondence to Debabrata Dutta, Raúl Padrón or Roger Craig.

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Extended data figures and tables

Extended Data Fig. 1 Cryo-EM imaging and data processing.

a, Cryo-EM image of human cardiac thick filament. b, 2D-class averages of 5-crown-long segments of C-zone. c, Enlargement of 2D class average. Two IHMs (CrH and CrT) show strong density while the other (CrD) is fuzzy, indicating mobility. d, 2D class average of M-line region, included in some images (3D reconstruction to be published separately). e, f, The two 5-crown reconstructions of the C-zone† produced by cryoSPARC (see Methods), showing local resolution (coloured maps, with resolution scale in Å at bottom), global resolution estimate (gold standard Fourier shell correlation (GSFSC) curves, upper), and heat maps of angles of view of particles used in the reconstruction (lower). The local resolution maps show best resolution in the central 3 crowns; disorder in CrD is indicated by its lower resolution (red) compared with CrH and CrT. Analysis of the maps (see text) shows highest resolution (blue) where interactions occur between components, which would stabilize these regions. The heat maps show a good distribution of angles of view. Most particles are close to in-plane (elevation axis), due to extended, relatively rigid nature of the thick filament, while all rotations about the filament axis are included, with a small preferred-orientation every 120° (darker patches along azimuth axis). †Particle picking for the reconstructions was automated (Methods). We assume that the particles originate from the C-zone because: 1. The C-zone is the longest head-bearing region of each half-filament and therefore will be the source of most particles. 2. The reconstructions are fully compatible with C-zone reconstructions where particles were user-selected from the C-zone17,18. 3. The reconstructions show an extended molecule that can only be interpreted as cMyBP-C. 4. The titin strands in the reconstruction exhibit an 11-domain super-repeat, providing strong evidence that the particles contributing to the structure come from the C-zone, while D-zone particles, with their shorter, 7-domain titin repeat, have been excluded.

Extended Data Fig. 2 Abbreviations and overview of interactions in 3D reconstruction.

a, Abbreviations and colour coding used to describe reconstruction and interactions in the thick filament. Top table shows correspondence between nomenclature and colour coding used in this paper and that used in the accompanying manuscript by Tamborrini et al.19. b, Interaction network between titin, myosin (heads and tails), and cMyBP-C. (1) titins (TA, TB) interact with each other (at 2 points only), with CrH and CrT tails (TA), and with CrH and CrD tails (TB), but not with cMyBP-C. (2) cMyBP-C interacts with CrH and CrT FHs, and possibly with CrD BH, with CrT and CrD tails, but not with titin. (3) all tails interact with each other (TaH-TaT, TaT-TaD, TaH-TaD) and with themselves (TaH-TaH, TaT-TaT, TaD-TaD). Neither titin, cMyBP-C, nor myosin (heads or tails) hold the thick filament together alone; instead, all orchestrate its interacting, asymmetric structure. c. Cartoon showing interactions in the reconstruction viewed transversely towards the Z-line, exhibiting three equivalent radial sectors I-III. Due to 3-fold symmetry, the positions of the dividing dotted red lines between sectors are arbitrary. We have chosen them to produce a division in which most interactions occur within a sector, and few between sectors (Extended Data Fig. 4a). The sectors thus defined may correspond to the 3 subfilaments into which vertebrate thick filaments fray at low ionic strength71, as these would break the fewest interactions. Only two interactions, between titins TB and TA, in neighbouring sectors, would be broken, at T1 and T8. Note how TB and TA pairs from adjoining sectors are nicely accommodated in the space between CrH and CrD IHMs (see also Fig. 1c,d). The structure suggests that when filaments are synthesized in the cell, three pre-formed sectors could assemble into a filament when TB and TA strands of different sectors zip together establishing the T1 and T8 interactions (Supplementary Discussion). A central core (grey, dashed circle) contains only CrT tails, interacting with themselves. d, Cartoon of full-length thick filament showing P-, C- and D-zones, bare zone (BZ) and M-line. Red, green and blue spheres represent CrD, CrH and CrT IHMs in 430-Å-long triplets (dotted box). Titin strands (yellow, orange) extend along filament. cMyBP-C (pink) extends longitudinally for part of its length, then projects out. e, Cartoon showing interactions in sector I of the reconstruction, as viewed from the bottom of c (i.e. from outside the filament). CrD, CrT and CrH IHMs comprise a single 430-Å-long triplet within the sector (dashed rectangle), with CrD and CrH from triplets of adjacent sectors (pale red and green ghosts) also shown. The dashed black lines in the three coloured IHMs denote the tilted angles of the CrD and CrT IHMs compared with the untilted (horizontal) IHM in CrH (tilt axis is a radial line extending from the filament axis through the centre of the IHM). CrD heads are dynamically disordered/mobile (curved double-arrows). In the reconstruction, cMyBP-C C5-C10 domains exhibit strong densities, while C2-C4 and C0-M, which are mobile, exhibit weaker (C2-C4, straight pink double arrows) or much weaker (C0-M; grey, curved double-arrows) densities, C0-M possibly docking intermittently on the CrD FH (green dotted fragment). In the sarcomere, the mobile C0-M domains may detach from the thick filament, extending out and binding to the thin filament (red dotted fragment41). Double oblique lines in tails indicate that only partial tails are shown. Red numbers of titin domains refer to the numbering in Fig. 4.

Extended Data Fig. 3 Validation of reconstruction.

a-d, We compared our reconstruction with X-ray diffraction data, which provide information on filament structure in the lattice of intact muscle. a, Longitudinal, and b-d, transverse views of our cardiac thick filament reconstruction at the 3 crowns in the 430 Å repeat (grey map), fitted with thick filament atomic model (CrH, green; CrT, blue; CrD, red) based on X-ray diffraction data22 combined with previous negative stain reconstructions17,18. The excellent fit of the X-ray model to the cryo-EM map, especially the radial positioning of the heads (centre-of-mass ~135 Å from the filament axis22), suggests that the reconstruction is close to the structure in intact muscle, and strongly supports its validity. The previous negative stain reconstructions had suggested a head centre-of-mass at ~95 Å radius (~40 Å less than the cryo-reconstruction), thought to be due to radial collapse of heads during drying of negative stain22—not an issue with frozen-hydrated specimens. e-f, We also compared an averaged power spectrum of the reconstruction (rotated at intervals of 10° from 0 to 110°) (e) with an averaged power spectrum of selected filaments used in the reconstruction (f). The similarity of the two power spectra, both extending to the 36th order of the 430 Å repeat (12 Å), supports the validity of the reconstruction. g, Wide-angle X-ray diffraction pattern of intact, relaxed porcine cardiac muscle at same scale (courtesy of Drs. Tom Irving and Weikang Ma, unpublished data) shows similar myosin layer lines at low angles, further suggesting that our structure is similar to that in intact muscle. A key feature of all patterns is the prominent 39 Å reflection (11th order of 430 Å, red arrows). Previous X-ray studies have suggested that this may be due to titin72. Our reconstruction reveals titin unambiguously (see text and Extended Data Fig. 5), and we show that the reflection arises from the kinking of its elongated structure, allowing 11 domains to fit into the 430 Å repeat (Extended Data Fig. 6c). h, i. We also compared our native filament IHMs (CrH and CrT) with the cryo-EM structure of a recombinant IHM containing 15 heptads of tail (PDB 8ACT46). 8ACT (yellow) was superimposed on CrH (green) and CrT (blue) (h, i, respectively). Superposition was performed by matching BHs (Matchmaker in ChimeraX) to reveal how the FH is positioned with respect to the BH in each case. The 3 different IHMs showed strong similarity. However, the two filament IHMs are non-identical (Fig. 2h), due to different interactions with cMyBP-C and the backbone in the environment of the native filament (Fig. 5c–f, Fig. 6g–m). Although the fit of 8ACT to the filament IHMs was not perfect, its individual blocked and free heads showed excellent correspondence to the respective CrT and CrH heads. CrH BH and 8ACT BH RMSD between 618 pruned atom pairs was 1.179 Å, and across all 867 pairs was 2.148 Å; CrH FH and 8ACT FH RMSD between 592 pruned atom pairs was 1.268 Å, and across all 851 pairs was 3.371 Å; CrT BH and 8ACT BH RMSD between 554 pruned atom pairs was 1.247 Å, and across all 867 pairs was 2.328 Å; CrT FH and 8ACT FH RMSD between 531 pruned atom pairs was 1.287 Å, and across all 851 pairs was 2.526 Å. We conclude that 8ACT is an excellent model for the two filament IHMs, its individual heads matching essentially perfectly with the individual free and blocked heads of CrT and CrH, and the whole IHM being close to the CrT and CrH whole IHMs (overall closer to CrH). The small difference between the isolated IHM and its counterparts in the filament is likely due to the absence in the isolated molecule of interactions of the FH and BH that occur with cMyBP-C and the filament backbone in the native filament. RMSD of Cα was calculated with the ChimeraX Matchmaker command using FH and BH separately.

Extended Data Fig. 4 Tracking myosin tails and their interactions (zoom for detail).

a, Transverse slices of reconstruction (looking towards M-line) showing the tail arrangement at crowns H, T, and D, respectively (coloured lines in c) for TaH (green), TaT (blue) and TaD (red). Tails are tracked by showing their positions at different crown levels (numerals), using the convention devised by Squire21,26, as they travel from their N-terminal origins, at the head-tail junction of each IHM (level 1), to their C-terminal tips (level 11). Black lines in coloured sectors replace the numerals, revealing the quite different courses of the 3 tail types as they travel along the filament. For example, TaD lie near the surface while TaT are the most central, forming the filament core. Titins A and B are labelled TAn and TBn, where n is the nth titin domain in the 430 Å repeat. MyBP-C is pink and labelled Cn (where n is the MyBP-C domain30). The dotted black lines show one sector (coloured) at each crown. Rectangles show examples of interactions of tails staggered by 1, 3, 5, and 7 crowns. Note sheets of CrD tails (red) staggered by 3 crowns (430 Å) near surface of backbone, forming a binding platform for cMyBP-C (see text). b, Cross-section at CrH, showing map (left) and corresponding space-filling atomic model (right), revealing clear examples of contact (thus interaction) between myosin tails with various staggers. Similar contacts are seen at other levels. c, Longitudinal view of map (M-line at right) showing where slices in a are cut. d-f, Longitudinal views of reconstruction showing interactions of tails staggered by 1, 3, 5 or 7 crowns (~143, 430, 715 or 1,001 Å), as predicted by23, corresponding to differences in tail numbers of 1, 3, 5, and 7 in a. Main sites of contact are inside the rectangular boxes. The most common sites are the distal LMM regions of each tail, including the  assembly competence domain (ACD) at the C-terminus (top boxes), consistent with its requirement for filament assembly (see text). The most prevalent stagger (3-crowns, 430 Å) is confined to homologous tails (TaH-TaH, TaT-TaT, TaD-TaD) (e). Heterologous tail combinations are staggered by 5 crowns (TaT-TaD, TaD-TaH) and one (TaH-TaD) by 7 crowns (f). One heterologous pair (TaH-TaT) exhibits a 141 Å stagger between S2s in the same sector (d, right) and between LMMs (including the ACD) from different sectors (d, left). g, Examples of charge interactions between S2’s staggered by 141 Å and LMMs by 430 Å (red, negative; blue, positive). h, Map and model at the 4 skip residues in CrH tails (Supplementary Fig. 3) and comparison of skip 1 in the filament (green) with corresponding crystal structure of skip 1 (PDB 4xa1, pink24). The similarity gives support for the relevance of this X-ray model to the native structure. i, CrH, CrT and CrD tails with skip residues marked. All skips in the 3 tails appear to be associated with a longer coiled-coil pitch (~100–110 Å compared with regular ~75 Å) and with bends in the tail. Interestingly, the bends are not identical for all three tail types. Skip 1 of TaT and TaH shows a slight bend, while the TaD bend is sharper; skip 2 of TaT shows a slight bend, while TaD and TaH bends are sharp; skip 3 of all tails shows a mildly bent structure; and skip 4 bend is stronger in TaT and TaD than TaH. See also Supplementary Videos 2 and 3.

Extended Data Fig. 5 Domain identification and model building of titins and cMyBP-C (zoom for detail).

a, e. AlphaFold65-predicted models of the two titins (a) and cMyBP-C (e). The sequence for titin super-repeat 4 of the C-zone was used, as each super-repeat is unique. Individual domains of titin and cMyBP-C showed high confidence. pLDDT (predicted local distance difference test) was ~90 (high confidence; dark blue in a). b, f, Predicted aligned error (PAE) was also good (<5 Å). From PAE plots, portions where the conformation of inter-domain linkers also had high confidence (yellow and pink boxes) were initially rigid-body fitted to the reconstruction (‘fit in map’ in ChimeraX), and other domains then connected using Coot and flexible fitting (MDFF, NAMDINATOR) to build the final atomic models (Methods). c, g, Atomic models (“Final Model”) of TB, TA and cMyBP-C. Simulated maps (“Molmap”: yellow, orange, pink surfaces) were generated from the models, using Molmap in ChimeraX, to compare with the EM density in the reconstruction (grey surface maps segmented from full reconstruction). The simulated maps showed excellent agreement with the actual map for every domain, giving confidence in our atomic models for the two titins and cMyBP-C. An especially striking prediction of AlphaFold was the long linker (e) between cMyBP-C domains C9 and C10 (g), which fitted precisely into our EM map. d, h, Predicted density maps (yellow, orange, pink), based on atomic models (c, g), placed on the full reconstruction (grey), for comparison in the context of the entire map (actual densities outlined by yellow, orange and pink borders). The similarity of the actual and generated maps confirms our confidence in the assignment of cMyBP-C and titin domains and their near-atomic structures. See Supplementary Methods, Supplementary Figs. 1, 2, and Supplementary Video 4 for additional information on fitting of titin domains.

Extended Data Fig. 6 Different conformations of titins TB and TA (zoom for detail).

a, The two titin densities (TB, TA) in one 430-Å super-repeat of a sector contain 11 Ig and Fn domains, and their overall conformation is not straight. The titin maps segmented from the reconstruction show that, due to the presence of 3 kinks (arrows), and a slight curve in the T1-T3 and T8-T11 regions, one super-repeat (11 domains) fits precisely into 430 Å, matching exactly the triplet repeat of myosin heads (CrD-CrT-CrH). The kinks, all occurring at an Ig-Fn junction, demarcate three parts of the super-repeat: 1. T1-T3 (Ig-Fn-Fn); 2. T4-T7 (Ig-Fn-Fn-Fn); and 3. T8-T11 (Ig-Fn-Fn-Fn). Parts 1 and 3 of TB and TA have similar conformations, and are approximately parallel to each other. However, the conformation of Part 2 differs between TB and TA. In TB, T4-T7 is rotated ~90° with respect to TA, positioning it at a higher radius above the surface of the backbone, where it becomes available for interaction with CrD S2 as shown in b (see also Supplementary Video 1). b, Because of this conformational difference, the middle portions of TB and TA interact differently with different tails. The bent and raised conformation of T5-T6-T7 in TB binds to proximal S2 of CrD with mostly electrostatic attraction (inset), while the straight conformation of T5-T6 in TA binds to distal S2 of CrH (Extended Data Fig. 7), generating an axial shift of 148 Å between CrH and CrD (see d). c, Previous studies speculated that the 39 Å reflection in the X-ray diffraction pattern of relaxed muscle (Extended Data Fig. 3g) may be due to titin72. Here we demonstrate this directly. Fast Fourier transformation (FFT) of the titin strand, segmented from the reconstruction in its native (kinked) conformation (right), produces a meridional reflection at a spacing of 39 Å, the 11th order of the 430 Å repeat (seen as layer lines at lower angles). If titin is computationally straightened (left), the reflection moves towards the origin (~44 Å spacing), producing a reflection that is not observed in the relaxed X-ray pattern. Agreement of the reconstruction with X-ray data from relaxed intact muscle (Extended Data Fig. 3g vs. e, f) implies that titin is kinked in the native state, allowing the 11 domains of its super-repeat to fit into 430 Å. The FFTs were computed from 8 titin super-repeats of the kind shown in c, laid end-to-end, to create a strand similar to that in one C-zone of the thick filament. See also Extended Data Fig. 10. d, Past studies suggested that the uneven spacing of titin’s 3 Ig domains in the super-repeat might be responsible for the uneven spacing of the 3 myosin crowns in the 430 Å repeat. Our reconstruction enables us to test this idea. We find an approximate correlation between the positions of the 3 Ig domains (purple) and the motor domains of the 3 IHMs. Ig1 (T1) correlates with CrT, Ig2 (T4) with CrD, and Ig3 (T8) with CrH. But we see no precise correlation that would suggest that the 3 Ig domains directly position the crowns (see crown and Ig spacings on figure). This is not surprising, given the absence of any direct titin-head interaction in the structure. Instead, titin positions the crowns through interaction of TB and TA with CrH and CrD tails, while CrT is positioned by interaction of TaT with TaH (see text). See also Supplementary Video 5.

Extended Data Fig. 7 Titin-tail and tail-tail electrostatic interactions create the unique 3-crown repeat of myosin molecules in the C-zone (zoom for detail).

Myosin tails form extensive, mostly electrostatic, interactions with each other and with titin, which combined determine the axial and azimuthal positions of CrH, CrT, and CrD. a, b show representative examples. a, TaH and TaT (atomic models fitted into density map) run together in the S2 region (black rectangle) (Fig. 3), forming charge-charge interactions (right: red, negative; blue, positive), which shift CrT ~141 Å axially with respect to CrH (cf.23; see also Extended Data Fig. 4). b, CrH tails also form electrostatic interactions with TA, involving all 11 domains of the 430 Å titin super-repeat (Fig. 4c). Interactions involve both proximal and distal S2 (bottom and top right, respectively), whose ~42 Å charge repeat23 matches the ~42 Å spacing of the charged-surface titin domains. This is the most extensive titin-tail interaction in the reconstruction and is responsible for placing CrH crowns 430 Å apart, by matching them to the 430 Å length of the TA 11-domain super-repeat in its kinked conformation on the filament surface (Extended Data Fig. 6c). In summary, TaH-TA and TaH-TaT interactions (shown here), and TaD-TB interaction (Extended Data Fig. 6b), are the driving force for organizing the three crowns (CrH-CrT-CrD) in a quasi-helical arrangement in the human cardiac thick filament in the relaxed state.

Extended Data Fig. 8 cMyBP-C position is determined by binding to a specific coalescence of CrD tails (zoom for detail).

The reconstruction shows that C-terminal domains C6 to C10 of cMyBP-C dock onto the filament backbone through interaction with only one type of tail (CrD tails, TaD). There is no interaction with titin, although titin is indirectly involved by positioning the tails. The complete cMyBP-C binding site is formed when three CrD tails, from three consecutive 430 Å repeats, lie side-by-side, creating a sheet-like docking platform that can make complementary charge interactions with the cMyBP-C domains. a, Distribution of C6-C10 binding sites at different points along a CrD tail. Complete/partial binding sites are shown by pink circles/semicircles. b, When CrD tails from 3 levels assemble to form the docking platform, a complete binding site is created (green box). C6 and C7 interact with level 7 TaD LMM; C8 with level 1 S2; C9 with level 1 S2 and level 4 LMM; C9-C10 linker with level 4 LMM; and C10 with level 4 and level 7 LMMs. The docking of C6 to C10 on the backbone thus requires a particular arrangement of CrD tails, from levels 1, 4 and 7, 430 Å apart. Based on titin’s specific 11-domain super-repeat in the C-zone (determining myosin tail locations), and quite different 7-domain super-repeat in the D-zone27, this specific arrangement of CrD tails occurs only in the C-zone. This may explain cMyBP-C’s confinement only to this region of the thick filament. In addition to tail binding, C8 and C10 (both Ig domains) interact with the FH motor domain of CrH and CrT (Fig. 4). c, d, Atomic model fitted to map shows details of interactions in the complete cMyBP-C binding site on the 3 CrD tails. e, f, Surface charge depiction of tails (e) and cMyBP-C, rotated 180° (f), suggests that binding occurs mainly through electrostatic attraction (boxes show complementary charges). See also Supplementary Video 6.

Extended Data Fig. 9 Examples of mutations clustering at intermolecular interaction sites, whose location suggests novel mechanisms of HCM pathogenicity (zoom for detail).

The map reveals that many HCM pathogenic mutations in cMyBP-C, and in myosin tails and heads of specific crowns (CrH, CrT and CrD), are in intermolecular interfaces in the native filament. These mutations could not be mapped previously due to use of the tarantula filament IHM, lacking cMyBP-C and titin5,6. a, Mutations affecting the tail-titin interface. Mutations in ring 1 (D906G) and 2 (E924K, E930K) of CrD S248 occur at the site of interaction with TB domains T5 and T6. Surface charge depiction showing positively charged patches on TB (blue on right inset) suggests that these mutations, involving loss or reversal of negative charge, would weaken binding of CrD S2 to TB. This could interfere with transmission of tension by titin to the cMyBP-C–TaD binding site, and thus disrupt possible mechanical signalling mechanisms (see text and Extended Data Fig. 10). b, Mutations affecting tail-tail interfaces. Mutation E924K in (a) is also involved in a tail-tail interaction, in this case not CrD, but the S2s of CrH and CrT. E924K charge reversal on CrT S2 (right inset) would be expected to weaken interaction with positive charge (blue) on CrH S2, thus impairing stability of the tail network. Disruption of function in these 2 different ways (tail-TB and tail-tail) could make the E924K mutation especially pathogenic. c, Mutations in cMyBP-C and the myosin motor domain affecting the cMyBP-C–myosin head interface. Pathogenic mutations in the myosin head (P307H) and the cMyBP-C C8 domain (R1002Q, E1017K) both occur in the interaction interface of C8 with the FH motor domain of CrH. Surface charge depiction suggests that charge loss or reversal on C8 (left inset) and on CrH FH (right inset) would weaken this interface, impairing cMyBP-C’s stabilization of the CrH FH. This could disturb its SRX state and interfere with proposed mechanical signalling mechanisms (Extended Data Fig. 10). d, Mutations in the myosin motor domain affecting multiple interfaces with tails. The classic mutation R403Q, in the CM loop of the motor domain, has been widely studied but not fully explained73. The reconstruction suggests that it could have multiple effects, by impairing head-tail interactions differently in CrH and CrT FHs. In the upper inset, the CrT CM loop interacts with its own S2, while in the lower, the CrH CM loop interacts with S2 from a more distal CrT (cf. Fig. 6g-i). Loss of positive charge in the former may strengthen binding to the positive charge K939, stabilizing the IHM, while in the latter it would weaken interaction with the CrT tail (E1119/E1120), again affecting mechanical signalling mechanisms (Extended Data Fig. 10). In summary, due to the various environments of myosins in a single 430-Å repeat, a single mutation can affect multiple interactions, with different pathogenic consequences.

Extended Data Fig. 10 Proposed mechanism of length-dependent activation (LDA) and mechanosensing (MS) in cardiac thick filament involving titin, cMyBP-C and myosin tails (zoom for detail).

Our reconstruction reveals connections of titin to myosin and myosin to cMyBP-C, that may underlie LDA and MS. a, b. Upper (relaxed): In relaxed state (right), TB and TA are kinked as in our reconstruction (left). Myosin heads form IHMs; cMyBP-C binds to docking site on CrD tails (green box; Extended Data Fig. 8) and to CrH and CrT heads (asterisks) (Fig. 5c–f); and TB and TA interact with myosin tails (red, green connections). In addition, CrH FH CM loop binds to TaT tail (“+” in b (upper); Fig. 6g, h). Middle (LDA): Elongation of the sarcomere at end-diastole (right) stretches TB and TA, reducing the kinks (left), supported by an increase in the spacing of the 39 Å X-ray reflection upon stretch74; see Extended Data Figs. 3g, 6c). Tension-induced translation of titin domains pulls on connected myosin tails, partially dismantling cMyBP-C binding sites on CrD tails and on CrH and CrT heads. Loss of the stabilizing influence of cMyBP-C on CrH and CrT, augmented by weakening of the CrH FH CM loop-TaT interaction (“+” in b, upper) under tension, releases heads for actin interaction, accounting for progressive enhanced force of systole that follows corresponding sarcomere length increase (=LDA). Lower (MS): In mechanosensing (right), CrD heads that performed a “sentinel” role for detecting thin filament activation75,76 in relaxed and LDA states, are now active and produce tension by interacting with actin (right). This stretches titin in a similar way to LDA (left), with similar consequences, enhancing contractility. In addition to the above cMyBP-C interactions, there are also CrD tail interactions with CrH FH and CrT FH, which could stabilize these IHMs (Fig. 6j, k, m). These interactions would also be broken upon sliding of CrD tails, contributing to LDA and MS.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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Supplementary information

Supplementary Information

Supplementary Methods (cryo-EM workflow; fitting of titin domains to map), Supplementary Discussion (model for thick filament assembly), Supplementary Table 1 (list of sequences used), and Supplementary Figs. 1 (uniqueness of fit of Ig and Fn domains into cryo-EM map), 2 (rigid body fitting of titin domains to cryo-EM map of titin) and 3 (annotated myosin heavy chain sequence alignment).

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Supplementary Video 1

3D map fitted with refined atomic model. The map (EMD-29722) is fitted with the refined model (PDB: 8G4L). The map is rotated around the y axis and then becomes transparent and magnified to show the atomic fitting. Finally, the map disappears and the model is rotated 90° around the x axis to show the transverse view (Figs. 1 and 2). CrH: BH, green; FH, pale green; ELC, goldenrod; RLC, chocolate. CrT: BH, royal blue; FH, sky blue; ELC, salmon; RLC, dark violet. CrD: BH, red; FH, tomato. TA, dark orange; TB, yellow; cMyBP-C, deep pink.

Supplementary Video 2

Segmented maps of full-length myosin tails from CrH, CrT and CrD showing their different paths. The five-crown map is extended to 14 crowns (Methods) to track the CrH, CrT and CrD tails (Fig. 3). The extended map is rotated around the y axis and the organization of the tails is shown for one crown at a time (for clarity). CrH:TaH, green; CrT, TaT, royal blue; CrD: TaD, red.

Supplementary Video 3

The effect of skip residues in the myosin coiled-coil on the conformation of the tails. The full-length structure of each of the three types of myosin molecule (CrT, CrD, CrH) is shown in sequence. The molecules are rotated about their long axis to reveal in 3D the local conformational changes near the skip residues (yellow spheres). See also Extended Data Fig. 4i.

Supplementary Video 4

Uniqueness of fit of the Ig and Fn domains into TA and TB in the EM map. We fitted AlphaFold-predicted Ig domains into neighbouring Ig or Fn domains of TB (yellow) in our map, and similarly with Fn domains. Rotation of the titin strands demonstrates an excellent 3D fit into the map according to our interpretation (Fig. 2c,d and Extended Data Fig. 5a–d) and poor fit when the domains are swapped. We obtained the same result with TA (orange). See Supplementary Methods and Supplementary Fig. 1.

Supplementary Video 5

The organization of titin super-repeats in TB and TA with respect to full-length myosins from the three crowns. The extended map, shown in silhouette for simplicity, is rotated around the y axis. Segmented maps of TB and TA, and of CrH, CrT and CrD myosins appear in order (Fig. 4). CrH, TaH, green; CrT, TaT, royal blue; CrD, TaD, red; TA, dark orange; TB, yellow.

Supplementary Video 6

3D segmented map of cMyBP-C with interacting myosin heads and tails, fitted with the refined atomic model. The map is rotated around the y axis and then becomes transparent to show the atomic model with interacting interfaces (Fig. 5) of cMyBP-C domains C5 to C10 with CrH and CrT IHMs. c-MyBP-C docking sites on the sheet of 3 TaD tails (Extended Data Fig. 8) are also depicted. Finally, the map disappears and the model rotates around the y axis. CrH: BH, green; FH, pale green; ELC, goldenrod; RLC, chocolate. CrT: BH, royal blue; FH, sky blue; ELC, salmon; RLC, dark violet. TaD α-helices, red, tomato; cMyBP-C, deep pink.

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Dutta, D., Nguyen, V., Campbell, K.S. et al. Cryo-EM structure of the human cardiac myosin filament. Nature 623, 853–862 (2023). https://doi.org/10.1038/s41586-023-06691-4

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