Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Internal structure and visualization of transmembrane domains of the RyR1 calcium release channel by cryo-EM

Nature Structural & Molecular Biology volume 12, pages 539–544 (2005)Cite this article

Abstract

RyR1 is an intracellular calcium channel with a central role in muscle contraction. We obtained a three-dimensional reconstruction of the RyR1 in the closed state at a nominal resolution of ∼10 Å using cryo-EM. The cytoplasmic assembly consists of a series of interconnected tubular structures that merge into four columns that extend into the transmembrane assembly. The transmembrane assembly, which has at least six transmembrane α-helices per monomer, has four tilted rods that can be fitted with the inner helices of a closed K+ channel atomic structure. The rods splay out at the lumenal side and converge into a dense ring at the cytoplasmic side. Another set of four rods emerges from this ring and shapes the inner part of the four columns. The resulting constricted axial structure provides direct continuity between cytoplasmic and transmembrane assemblies, and a possible mechanism for control of channel gating through conformational changes in the cytoplasmic assembly.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Image processing.
Figure 2: Isosurface representation of the RyR1 in different views.
Figure 3: Internal structure of the cytoplasmic assembly and the columns.
Figure 4: Substructure of the transmembrane assembly.
Figure 5: Schematic of the architecture of the RyR.

Similar content being viewed by others

Accession codes

Accessions

BINDPlus

References

  1. Fill, M. & Copello, J.A. Ryanodine receptor calcium release channels. Physiol. Rev. 82, 893–922 (2002).

    Article  CAS  Google Scholar 

  2. Dulhunty, A.F. & Pouliquin, P. What we don't know about the structure of ryanodine receptor calcium release channels. Clin. Exp. Pharmacol. Physiol. 30, 713–723 (2003).

    Article  CAS  Google Scholar 

  3. Wagenknecht, T. Samsó, M. Three-dimensional reconstruction of ryanodine receptors. Front. Biosci. 7, d1464–d1474 (2002).

    Article  Google Scholar 

  4. Wolf, M., Eberhart, A., Glossmann, H., Striessnig, J. & Grigorieff, N. Visualization of the domain structure of an L-type Ca2+ channel using electron cryo-microscopy. J. Mol. Biol. 332, 171–182 (2003).

    Article  CAS  Google Scholar 

  5. Paolini, C., Protasi, F. & Franzini-Armstrong, C. The relative position of RyR feet and DHPR tetrads in skeletal muscle. J. Mol. Biol. 342, 145–153 (2004).

    Article  CAS  Google Scholar 

  6. Wagenknecht, T. et al. Locations of calmodulin and FK506-binding protein on the three-dimensional architecture of the skeletal muscle ryanodine receptor. J. Biol. Chem. 272, 32463–32471 (1997).

    Article  CAS  Google Scholar 

  7. Samsó, M. & Wagenknecht, T. Apocalmodulin and Ca2+-calmodulin bind to neighboring locations on the ryanodine receptor. J. Biol. Chem. 277, 1349–1353 (2002).

    Article  Google Scholar 

  8. Orlova, E.V. Serysheva, II, van Heel, M., Hamilton, S.L. & Chiu, W. Two structural configurations of the skeletal muscle calcium release channel. Nat. Struct. Biol. 3, 547–552 (1996).

    Article  CAS  Google Scholar 

  9. Sharma, M.R., Jeyakumar, L.H., Fleischer, S. & Wagenknecht, T. Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy. J. Biol. Chem. 275, 9485–9491 (2000).

    Article  CAS  Google Scholar 

  10. Radermacher, M. et al. Cryo-electron microscopy and three-dimensional reconstruction of the calcium release channel/ryanodine receptor from skeletal muscle. J. Cell Biol. 127, 411–423 (1994).

    Article  CAS  Google Scholar 

  11. Serysheva, I.I. & Hamilton, S.L., Chiu, W. & Ludtke, S.J. Structure of Ca2+ release channel at 14 Å resolution. J. Mol. Biol. 345, 427–431 (2005).

    Article  CAS  Google Scholar 

  12. Takeshima, H. et al. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 339, 439–445 (1989).

    Article  CAS  Google Scholar 

  13. Zorzato, F. et al. Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 265, 2244–2256 (1990).

    CAS  PubMed  Google Scholar 

  14. Grunwald, R. & Meissner, G. Lumenal sites and C terminus accessibility of the skeletal muscle calcium release channel (ryanodine receptor). J. Biol. Chem. 270, 11338–11347 (1995).

    Article  CAS  Google Scholar 

  15. Balshaw, D., Gao, L. & Meissner, G. Luminal loop of the ryanodine receptor: a pore-forming segment? Proc. Natl. Acad. Sci. USA 96, 3345–3347 (1999).

    Article  CAS  Google Scholar 

  16. Zhao, M. et al. Molecular identification of the ryanodine receptor pore-forming segment. J. Biol. Chem. 274, 25971–25974 (1999).

    Article  CAS  Google Scholar 

  17. Welch, W., Rheault, S., West, D.J. & Williams, A.J. A model of the putative pore region of the cardiac ryanodine receptor channel. Biophys. J. 87, 2335–2351 (2004).

    Article  CAS  Google Scholar 

  18. Du, G.G., Sandhu, B., Khanna, V.K., Guo, X.H. & MacLennan, D.H. Topology of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1). Proc. Natl. Acad. Sci. USA 99, 16725–16730 (2002).

    Article  CAS  Google Scholar 

  19. Wang, R. et al. The predicted TM10 transmembrane sequence of the cardiac Ca2+ release channel (ryanodine receptor) is crucial for channel activation and gating. J. Biol. Chem. 279, 3635–3642 (2004).

    Article  CAS  Google Scholar 

  20. Williams, A.J., West, D.J. & Sitsapesan, R. Light at the end of the Ca2+-release channel tunnel: structures and mechanisms involved in ion translocation in ryanodine receptor channels. Q. Rev. Biophys. 34, 61–104 (2001).

    Article  CAS  Google Scholar 

  21. Doyle, D.A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998).

    Article  CAS  Google Scholar 

  22. Rosenthal, P.B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  Google Scholar 

  23. Malhotra, A. et al. Escherichia coli 70 S ribosome at 15 Å resolution by cryo-electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein. J. Mol. Biol. 280, 103–116 (1998).

    Article  CAS  Google Scholar 

  24. Samsó, M., Trujillo, R., Gurrola, G.B., Valdivia, H.H. & Wagenknecht, T. Three-dimensional location of the imperatoxin A binding site on the ryanodine receptor. J. Cell Biol. 146, 493–499 (1999).

    Article  Google Scholar 

  25. Jiang, W., Baker, M.L., Ludtke, S.J. & Chiu, W. Bridging the information gap: computational tools for intermediate resolution structure interpretation. J. Mol. Biol. 308, 1033–1044 (2001).

    Article  CAS  Google Scholar 

  26. Fujiyoshi, Y. The structural study of membrane proteins by electron crystallography. Adv. Biophys. 35, 25–80 (1998).

    Article  CAS  Google Scholar 

  27. Block, B.A., Imagawa, T., Campbell, K.P. & Franzini-Armstrong, C. Structural evidence for direct interaction between the molecular components of the transverse tubule/sarcoplasmic reticulum junction in skeletal muscle. J. Cell Biol. 107, 2587–2600 (1988).

    Article  CAS  Google Scholar 

  28. Wagenknecht, T., Hsieh, C.E., Rath, B.K., Fleischer, S. & Marko, M. Electron tomo-graphy of frozen-hydrated isolated triad junctions. Biophys. J. 83, 2491–2501 (2002).

    Article  CAS  Google Scholar 

  29. Meissner, G. Ryanodine activation and inhibition of the Ca2+ release channel of sarcoplasmic reticulum. J. Biol. Chem. 261, 6300–6306 (1986).

    CAS  PubMed  Google Scholar 

  30. Unwin, N. Nicotinic acetylcholine receptor at 9 Å resolution. J. Mol. Biol. 229, 1101–1124 (1993).

    Article  CAS  Google Scholar 

  31. Pessah, I.N. & Zimanyi, I. Characterization of multiple [3H]ryanodine binding sites on the Ca2+ release channel of sarcoplasmic reticulum from skeletal and cardiac muscle: evidence for a sequential mechanism in ryanodine action. Mol. Pharmacol. 39, 679–689 (1991).

    CAS  PubMed  Google Scholar 

  32. Welch, W. et al. Structural components of ryanodine responsible for modulation of sarcoplasmic reticulum calcium channel function. Biochemistry 36, 2939–2950 (1997).

    Article  CAS  Google Scholar 

  33. Carroll, S., Skarmeta, J.G., Yu, X., Collins, K.D. & Inesi, G. Interdependence of ryanodine binding, oligomeric receptor interactions, and Ca2+ release regulation in junctional sarcoplasmic reticulum. Arch. Biochem. Biophys. 290, 239–247 (1991).

    Article  CAS  Google Scholar 

  34. Fessenden, J.D. et al. Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine. Proc. Natl. Acad. Sci. USA 98, 2865–2870 (2001).

    Article  CAS  Google Scholar 

  35. Kobayashi, S., Yamamoto, T., Parness, J. & Ikemoto, N. Antibody probe study of Ca2+ channel regulation by interdomain interaction within the ryanodine receptor. Biochem. J. 380, 561–569 (2004).

    Article  CAS  Google Scholar 

  36. Shtifman, A. et al. Interdomain interactions within ryanodine receptors regulate Ca2+ spark frequency in skeletal muscle. J. Gen. Physiol. 119, 15–32 (2002).

    Article  CAS  Google Scholar 

  37. Jiang, Y. et al. Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417, 515–522 (2002).

    Article  CAS  Google Scholar 

  38. Wagenknecht, T. et al. Cryoelectron microscopy resolves FK506-binding protein sites on the skeletal muscle ryanodine receptor. Biophys. J. 70, 1709–1715 (1996).

    Article  CAS  Google Scholar 

  39. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).

    Article  CAS  Google Scholar 

  40. Mindell, J.A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  Google Scholar 

  41. Grigorieff, N. Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase (complex I) at 22 Å in ice. J. Mol. Biol. 277, 1033–1046 (1998).

    Article  CAS  Google Scholar 

  42. Quillin, M.L. & Matthews, B.W. Accurate calculation of the density of proteins. Acta Crystallogr. D 56, 791–794 (2000).

    Article  CAS  Google Scholar 

  43. Huang, C.C., Couch, G.S., Pettersen, E.F. & Ferrin, T.E. Chimera: an extensive molecular modeling application constructed using standard components. Pac. Symp. Biocomput. 1, 724 (1996).

    Google Scholar 

Download references

Acknowledgements

We thank T. Walz for his generous sharing of the microscopy and image processing facilities at the Department of Cell Biology at Harvard Medical School, and for helpful discussions. We also acknowledge N. Grigorieff for allowing the use of CTFTILT and FREALIGN software, and the Resource for Visualization of Biological Complexity (US National Institutes of Health (NIH) RR01219) of Wadsworth Center for the use of the cryo-plunger apparatus. We thank Y. Cheng for his helpful discussions, advice with data analysis and technical support with the electron microscope, J. Frank for helpful discussions and X. Shen and J. Berkowitz for help with the RyR1 purification. This work was supported by RO1 AR43140 and PO1 AR17605 (to P.D.A.), AHA 0530147N (to M.S.), and AR40615 (to T.W.). The molecular EM facility at Harvard Medical School was established by a generous donation from the Giovanni Armenise Harvard Center for Structural Biology and is maintained by funds from NIH-GM62580 (to T. Walz).

Author information

Authors and Affiliations

  1. Brigham and Women's Hospital, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Montserrat Samsó & P D Allen

  2. New York State Department of Health, Wadsworth Center, Albany, 12201-0509, New York, USA

    Terence Wagenknecht

Authors
  1. Montserrat Samsó
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Terence Wagenknecht
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P D Allen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Montserrat Samsó.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Samsó, M., Wagenknecht, T. & Allen, P. Internal structure and visualization of transmembrane domains of the RyR1 calcium release channel by cryo-EM. Nat Struct Mol Biol 12, 539–544 (2005). https://doi.org/10.1038/nsmb938

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb938

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing