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.

Architecture and conformational switch mechanism of the ryanodine receptor

Abstract

Muscle contraction is initiated by the release of calcium (Ca2+) from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors (RyRs). RyRs are homotetrameric channels with a molecular mass of more than 2.2 megadaltons that are regulated by several factors, including ions, small molecules and proteins. Numerous mutations in RyRs have been associated with human diseases. The molecular mechanism underlying the complex regulation of RyRs is poorly understood. Using electron cryomicroscopy, here we determine the architecture of rabbit RyR1 at a resolution of 6.1 Å. We show that the cytoplasmic moiety of RyR1 contains two large α-solenoid domains and several smaller domains, with folds suggestive of participation in protein–protein interactions. The transmembrane domain represents a chimaera of voltage-gated sodium and pH-activated ion channels. We identify the calcium-binding EF-hand domain and show that it functions as a conformational switch allosterically gating the channel.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Architecture of RyR1.
Figure 2: Similarity between the architecture of RyR1 membrane domain and other ion channels.
Figure 3: Calcium-induced conformational changes in RyR1.
Figure 4: Mechanism of calcium release.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The coordinates and electron microscopy density maps for closed and open states have been deposited in the RCSB Protein Data Bank under the accession codes 4UWA and 4UWE, and in the Electron Microscopy Data Bank under the accession codes EMD-2751 and EMD-2752, respectively.

References

  1. 1

    Schwaller, B. in Calcium Signaling (ed. Islam, M. S. ) Ch. 1 1–25 (Springer, 2012)

  2. 2

    Baylor, S. M. & Hollingworth, S. Sarcoplasmic reticulum calcium release compared in slow-twitch and fast-twitch fibres of mouse muscle. J. Physiol. (Lond.) 551, 125–138 (2003)

    CAS  Google Scholar 

  3. 3

    Lanner, J. T., Georgiou, D. K., Joshi, A. D. & Hamilton, S. L. Ryanodine receptors: structure, expression, molecular details, and function in calcium release. Cold Spring Harb. Perspect. Biol. 2, a003996 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Kimlicka, L. & Van Petegem, F. The structural biology of ryanodine receptors. Sci. China. Life Sci. 54, 712–724 (2011)

    CAS  PubMed  Google Scholar 

  5. 5

    Hamilton, S. L. & Serysheva, I. I. Ryanodine receptor structure: progress and challenges. J. Biol. Chem. 284, 4047–4051 (2009)

    CAS  PubMed  Google Scholar 

  6. 6

    Van Petegem, F. Ryanodine receptors: allosteric ion channel giants. J. Mol. Biol. http://dx.doi.org/10.1016/j.jmb.2014.08.004 (15 August 2014)

  7. 7

    Parys, J. B. & De Smedt, H. Inositol 1,4,5-trisphosphate and its receptors. Adv. Exp. Med. Biol. 740, 255–279 (2012)

    CAS  PubMed  Google Scholar 

  8. 8

    Mackrill, J. J. Ryanodine receptor calcium channels and their partners as drug targets. Biochem. Pharmacol. 79, 1535–1543 (2010)

    CAS  PubMed  Google Scholar 

  9. 9

    Betzenhauser, M. J. & Marks, A. R. Ryanodine receptor channelopathies. Pflugers Arch. 460, 467–480 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Gulbis, J. M. & Doyle, D. A. Potassium channel structures: do they conform? Curr. Opin. Struct. Biol. 14, 440–446 (2004)

    CAS  PubMed  Google Scholar 

  11. 11

    Tung, C. C., Lobo, P. A., Kimlicka, L. & Van Petegem, F. The amino-terminal disease hotspot of ryanodine receptors forms a cytoplasmic vestibule. Nature 468, 585–588 (2010)

    ADS  CAS  Google Scholar 

  12. 12

    Sharma, P. et al. Structural determination of the phosphorylation domain of the ryanodine receptor. FEBS J. 279, 3952–3964 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Yuchi, Z., Lau, K. & Van Petegem, F. Disease mutations in the ryanodine receptor central region: crystal structures of a phosphorylation hot spot domain. Structure 20, 1201–1211 (2012)

    CAS  Google Scholar 

  14. 14

    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)

    ADS  CAS  PubMed  Google Scholar 

  15. 15

    Marcotte, E. M., Pellegrini, M., Yeates, T. O. & Eisenberg, D. A census of protein repeats. J. Mol. Biol. 293, 151–160 (1999)

    CAS  PubMed  Google Scholar 

  16. 16

    Huang, X., Fruen, B., Farrington, D. T., Wagenknecht, T. & Liu, Z. Calmodulin-binding locations on the skeletal and cardiac ryanodine receptors. J. Biol. Chem. 287, 30328–30335 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Maximciuc, A. A., Putkey, J. A., Shamoo, Y. & Mackenzie, K. R. Complex of calmodulin with a ryanodine receptor target reveals a novel, flexible binding mode. Structure 14, 1547–1556 (2006)

    CAS  PubMed  Google Scholar 

  18. 18

    Ponting, C., Schultz, J. & Bork, P. SPRY domains in ryanodine receptors (Ca2+-release channels). Trends Biochem. Sci. 22, 193–194 (1997)

    CAS  PubMed  Google Scholar 

  19. 19

    Liu, Z., Zhang, J., Wang, R., Wayne Chen, S. R. & Wagenknecht, T. Location of divergent region 2 on the three-dimensional structure of cardiac muscle ryanodine receptor/calcium release channel. J. Mol. Biol. 338, 533–545 (2004)

    CAS  PubMed  Google Scholar 

  20. 20

    Perfetto, L. et al. Exploring the diversity of SPRY/B30.2-mediated interactions. Trends Biochem. Sci. 38, 38–46 (2013)

    CAS  PubMed  Google Scholar 

  21. 21

    Kobe, B. & Kajava, A. V. When protein folding is simplified to protein coiling: the continuum of solenoid protein structures. Trends Biochem. Sci. 25, 509–515 (2000)

    CAS  PubMed  Google Scholar 

  22. 22

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

    ADS  CAS  PubMed  Google Scholar 

  23. 23

    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 

  24. 24

    Ferguson, D. G., Schwartz, H. W. & Franzini-Armstrong, C. Subunit structure of junctional feet in triads of skeletal muscle: a freeze-drying, rotary-shadowing study. J. Cell Biol. 99, 1735–1742 (1984)

    CAS  PubMed  Google Scholar 

  25. 25

    Yin, C. C., Blayney, L. M. & Lai, F. A. Physical coupling between ryanodine receptor-calcium release channels. J. Mol. Biol. 349, 538–546 (2005)

    CAS  PubMed  Google Scholar 

  26. 26

    Fessenden, J. D., Feng, W., Pessah, I. N. & Allen, P. D. Mutational analysis of putative calcium binding motifs within the skeletal ryanodine receptor isoform, RyR1. J. Biol. Chem. 279, 53028–53035 (2004)

    CAS  PubMed  Google Scholar 

  27. 27

    Lewit-Bentley, A. & Rety, S. EF-hand calcium-binding proteins. Curr. Opin. Struct. Biol. 10, 637–643 (2000)

    CAS  PubMed  Google Scholar 

  28. 28

    Kuboniwa, H. et al. Solution structure of calcium-free calmodulin. Nature Struct. Biol. 2, 768–776 (1995)

    CAS  PubMed  Google Scholar 

  29. 29

    Gao, L., Tripathy, A., Lu, X. & Meissner, G. Evidence for a role of C-terminal amino acid residues in skeletal muscle Ca2+ release channel (ryanodine receptor) function. FEBS Lett. 412, 223–226 (1997)

    CAS  PubMed  Google Scholar 

  30. 30

    Payandeh, J., Scheuer, T., Zheng, N. & Catterall, W. A. The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Tang, L. et al. Structural basis for Ca2+ selectivity of a voltage-gated calcium channel. Nature 505, 56–61 (2014)

    ADS  PubMed  Google Scholar 

  32. 32

    Liao, M., Cao, E., Julius, D. & Cheng, Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504, 107–112 (2013)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Uysal, S. et al. Mechanism of activation gating in the full-length KcsA K+ channel. Proc. Natl Acad. Sci. USA 108, 11896–11899 (2011)

    ADS  CAS  PubMed  Google Scholar 

  34. 34

    Ludtke, S. J. et al. Flexible architecture of IP3R1 by Cryo-EM. Structure 19, 1192–1199 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Smith, J. S. et al. Purified ryanodine receptor from rabbit skeletal muscle is the calcium-release channel of sarcoplasmic reticulum. J. Gen. Physiol. 92, 1–26 (1988)

    ADS  CAS  PubMed  Google Scholar 

  36. 36

    Samsó, M., Shen, X. & Allen, P. D. Structural characterization of the RyR1–FKBP12 interaction. J. Mol. Biol. 356, 917–927 (2006)

    PubMed  Google Scholar 

  37. 37

    Fischer, N., Konevega, A. L., Wintermeyer, W., Rodnina, M. V. & Stark, H. Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy. Nature 466, 329–333 (2010)

    ADS  CAS  PubMed  Google Scholar 

  38. 38

    Sitsapesan, R. & Williams, A. J. Gating of the native and purified cardiac SR Ca2+-release channel with monovalent cations as permeant species. Biophys. J. 67, 1484–1494 (1994)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Gaburjakova, M. et al. FKBP12 binding modulates ryanodine receptor channel gating. J. Biol. Chem. 276, 16931–16935 (2001)

    CAS  Google Scholar 

  40. 40

    Ahern, G. P., Junankar, P. R. & Dulhunty, A. F. Single channel activity of the ryanodine receptor calcium release channel is modulated by FK-506. FEBS Lett. 352, 369–374 (1994)

    CAS  PubMed  Google Scholar 

  41. 41

    Hawkes, M. J., Diaz-Munoz, M. & Hamilton, S. L. A procedure for purification of the ryanodine receptor from skeletal muscle. Membr. Biochem. 8, 133–145 (1989)

    CAS  PubMed  Google Scholar 

  42. 42

    Ritchie, T. K. et al. Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Campbell, M. G. et al. Movies of ice-embedded particles enhance resolution in electron cryo-microscopy. Structure 20, 1823–1828 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

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

    PubMed  Google Scholar 

  45. 45

    Chen, J. Z. & Grigorieff, N. SIGNATURE: a single-particle selection system for molecular electron microscopy. J. Struct. Biol. 157, 168–173 (2007)

    CAS  PubMed  Google Scholar 

  46. 46

    Hohn, M. et al. SPARX, a new environment for Cryo-EM image processing. J. Struct. Biol. 157, 47–55 (2007)

    CAS  PubMed  Google Scholar 

  47. 47

    Scheres, S. H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nature Methods 9, 853–854 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10, 584–590 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Penczek, P. A., Kimmel, M. & Spahn, C. M. Identifying conformational states of macromolecules by eigen-analysis of resampled cryo-EM images. Structure 19, 1582–1590 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nature Methods 11, 63–65 (2014)

    CAS  PubMed  Google Scholar 

  52. 52

    Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    PubMed  Google Scholar 

  53. 53

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Google Scholar 

  54. 54

    Pintilie, G. & Chiu, W. Comparison of Segger and other methods for segmentation and rigid-body docking of molecular components in cryo-EM density maps. Biopolymers 97, 742–760 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Pettersen, E. F. et al. UCSF chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    CAS  Google Scholar 

  56. 56

    Adamczak, R., Porollo, A. & Meller, J. Accurate prediction of solvent accessibility using neural networks-based regression. Proteins 56, 753–767 (2004)

    CAS  PubMed  Google Scholar 

  57. 57

    Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

    CAS  PubMed  Google Scholar 

  58. 58

    Fiser, A., Do, R. K. & Sali, A. Modeling of loops in protein structures. Protein Sci. 9, 1753–1773 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Kim, D. E., Chivian, D. & Baker, D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 32, W526–W531 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Kelley, L. A. & Sternberg, M. J. Protein structure prediction on the Web: a case study using the Phyre server. Nature Protocols 4, 363–371 (2009)

    CAS  PubMed  Google Scholar 

  61. 61

    Dosztányi, Z., Csizmok, V., Tompa, P. & Simon, I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21, 3433–3434 (2005)

    PubMed  Google Scholar 

  62. 62

    Tusnády, G. E. & Simon, I. The HMMTOP transmembrane topology prediction server. Bioinformatics 17, 849–850 (2001)

    PubMed  Google Scholar 

  63. 63

    Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    Pellegrini-Calace, M., Maiwald, T. & Thornton, J. M. PoreWalker: a novel tool for the identification and characterization of channels in transmembrane proteins from their three-dimensional structure. PLOS Comput. Biol. 5, e1000440 (2009)

    ADS  PubMed  PubMed Central  Google Scholar 

  65. 65

    DeLano, W. L. The PyMOL Molecular Graphics System version 1.3r1 (Schrödinger, LLC, 2010)

  66. 66

    Leitner, A. et al. Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography. Mol. Cell. Proteomics 11, M111.014126 (2012)

    PubMed  PubMed Central  Google Scholar 

  67. 67

    Leitner, A., Walzthoeni, T. & Aebersold, R. Lysine-specific chemical cross-linking of protein complexes and identification of cross-linking sites using LC–MS/MS and the xQuest/xProphet software pipeline. Nature Protocols 9, 120–137 (2014)

    CAS  PubMed  Google Scholar 

  68. 68

    Rinner, O. et al. Identification of cross-linked peptides from large sequence databases. Nature Methods 5, 315–318 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69

    Walzthoeni, T. et al. False discovery rate estimation for cross-linked peptides identified by mass spectrometry. Nature Methods 9, 901–903 (2012)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to O. Hofnagel for assistance with electron microscopy facilities and R. S. Goody for continuous support and useful comments on the manuscript. R.G.E. thanks R. Chaves and K. Willegems for assistance with particle selection. We gratefully acknowledge R. Matadeen and S. de Carlo (FEI Company) for image acquisition at the National Center for Electron Nanoscopy in Leiden (NeCEN) which is co-financed by grants from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (project 175.010.2009.001) and by the European Union’s Regional Development Fund through ‘Kansen voor West’ (project 21Z.014). This work was funded by Humboldt Foundation (to R.G.E.), by the ‘Deutsche Forschungsgemeinschaft’ Grant RA 1781/1-1 (to S.R.), the Max Planck Society (to S.R. and R.G.E.), VIB and Vrij Universiteit Brussel (to R.G.E.).

Author information

Affiliations

Authors

Contributions

R.G.E. designed the project and performed research, S.R. and R.G.E. managed the project, analysed data and wrote the manuscript, A.L. and R.A. performed cross-linking mass spectrometry experiments in the laboratory of R.A.

Corresponding authors

Correspondence to Rouslan G. Efremov or Stefan Raunser.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Quality of electron microscopy data.

aj, Data collected on a FEI Titan Krios in 1 mM EGTA (ae) and on a JEOL 3200 microscope in 10 mM CaCl2 (fj). a, f, Typical low dose cryo-EM micrographs of RyR1 vitrified in holey carbon grid recorded at an accelerating voltage of 300 kV (a) and 200 kV (f). b, g, Power spectra of the low dose micrographs. Thon rings are visible up to a resolution of 5 Å (b) and 10 Å (g). c, h, Representative two-dimensional class averages calculated in SPARX (c) and RELION (h) show characteristic views of RyR1. d, i, FSC curves between two independently refined half data sets (gold standard) shown for a set of all particles (solid black) and different conformational states (coloured curves). FSC between the volume calculated from all particles and the molecular model of RyR1 are shown as dotted black curves. FSC levels of 0.143 and 0.5 corresponding to the resolution cut off for density maps and the model are indicated. According to the 0.5 criterion the resolution of the model is 8.0 and 9.0 Å for the RyR1 reconstructions of closed and open states. The resolution is slightly lower than that of the map because of incompleteness of the model (the polyalanine model accounts for only about half of all the atoms of RyR1). e, j, Three-dimensional map of RyR1 coloured according to the local resolution calculated in ResMap from two unmasked volumes calculated from independently refined halves of the data sets. The resolution of the electron micoscropy map is heterogeneous with the highest resolution close to the centre of the molecule and the lowest resolution at the periphery. k, Statistics of single particle data.

Extended Data Figure 2 Consensus secondary structure prediction for rabbit RyR1 generated in SABLE.

Extension of domains is indicated by rectangles colour-coded as in Fig. 1. Except for the N-terminal AB domain and the three SPRY domains located in the first 1,650 residues, RyR1 is predicted to fold mainly in α-helices connected with coiled structures. The 165 amino acid sequence between residues 4,375 and 4,540, connecting the amphipathic region with the transmembrane domain, is glycine- and proline-rich and predicted to be disordered.

Extended Data Figure 3 Positions of transmembrane helices.

a, The probability for the formation of transmembrane helices calculated by the program TMHMM is shown for the C-terminal region of RyR1. Red bars indicate α-helices predicted in SABLE. The consensus transmembrane (TM) helices are unambiguously recognized and clearly separated, except of TM3 and TM4, which have nearly no solvent exposed loop in between. The amphipathic and pore helices are also clearly indicated, but have lower hydrophobicity scores than the transmembrane helices. b, Experimental mapping and prediction of transmembrane helices. Sequence ranges of computationally predicted, annotated and experimentally mapped transmembrane helices. Consensus helices between experimental mapping and computational prediction are indicated in bold. Although computational methods suggest just one TM3 helix instead of TM3/4, the width of the hydrophobic region corresponding to helix TM3 is consistent with the presence of two joint transmembrane helices as observed in the topology mapping experiments14.

Extended Data Figure 4 Cross-links identified within RyR1.

a, b, Graphical representation (a) and table (b) summarizing cross-links identified in cross-linking mass spectrometry experiments. a, Schematic representation of RyR1 domains colour-coded similar to domains in Fig. 1. The repeat 3–4 and EF-hand domains are shown as inserts into the α-solenoid 1.1 and α-solenoid 1.2 domains, respectively. The cross-links are shown by red lines. The approximate length of linkers joining domains is indicated by ‘Δ’ followed by the number of residues. Disordered regions of linking fragments are shown as fat dotted lines and their positions in the amino acid sequence are indicated. Domains containing β-sheets are depicted as circles, α-helical domains are shown as squares. b, Cross-linked residues are shown with ID score and their distances in the built model (see Supplementary Table 1 for complete list of identified cross-links).

Extended Data Figure 5 Cryo-EM density map of RyR1 at 6.1 Å resolution.

af, Backbone of the model in ribbon representation coloured as in Fig. 1 and cryo-EM density map from different regions of the structure are shown. a, Transmembrane region showing the inner helix and the helices of the voltage-sensor-like domain. The cytoplasmic part of the inner helix (in red) is resolved as good as the transmembrane helices indicating that the resolution inside the lipid nanodisc is similar to the nearby cytoplasmic regions. b, Pore helix and pore loops showing the absence of the second pore helix found in structurally homologous Nav channels. c, d, Helices of α-solenoid domains 1 (c) and 2 (d). e, Fit of the SPRY2 domain in the density. f, One of the least resolved domains, repeat 1–2, rod-shaped densities corresponding to α-helices are visible. g, Structure and density maps of individual domains. Segments of the 6.1 Å RyR1 density map corresponding to individual domains of RyR1 are shown together with the ribbon models of the domains in rainbow colouring from the N terminus (blue) to the C terminus (red). The excellent fitting of the crystallographic model of the N-terminal AB domain (residues 12–532)11 into the density, even revealing conformation of loops that were disordered in the crystal structure, indicates the high quality of our electron microscopy map. The density segment for FKBP bound to RyR1 was extracted from the 8.5 Å cryo-EM map of RyR1 in its open state.

Extended Data Figure 6 Details of the RyR1 structure.

a, The α-helical protrusion in α-solenoid 1 (contoured) includes an 80-Å-long α-helix and a bundle of helices exposed to solvent and represents a potential CaM-binding site. b, The phosphorylation loop (shown schematically since it was not resolved in the crystal structure) of repeat 3–4 (shown in rainbow colouring from N to C terminus) is exposed to solvent. Mutations of seven residues (four of which are shown as blue spheres) close to the potential phosphorylation site cause human diseases suggesting functional importance of this region and its potential involvement in the interaction with Cav1.1 channels. c, FKBP (red) bound to RyR1 resolved in the cryo-EM map of RyR1 in presence of 10 mM Ca2+. It interacts with the SPRY2 and the α-solenoid 1 domains. d, e, Comparison of the backbone of the RyR1 (coloured in red, yellow, green and blue) membrane domain with the TRPV1 channel (grey). Views perpendicular to (d) and parallel to (e) the membrane plane are shown. The tilt of transmembrane helices in the voltage-sensor domain (S1–S4) is different between the two proteins, while positions of inner helices (S6) in the gate region and architecture of the pore region shown as insert in e are similar.

Extended Data Figure 7 Conserved architecture of RyR and IP3R channels.

a, Conserved core between RyRs and IP3Rs. The evolutionarily most conserved and likely functionally important core includes part of the AB domain bridging the C-terminal regions of the α-solenoid 1 (blue), the C terminus of α-solenoid 1 (orange), which is in direct contact with the C-terminal domain (red). To indicate better the extent of the C-terminal domain the corresponding density segments are shown. The helices of the membrane domain are shown in dark blue. In the side view the density of the nanodisc is shown in light grey. The colour coding is identical to Fig. 1. b, c Fit of RyR1 domains in the IP3R1 density map. b, Rigid body fit of the RyR1 domains conserved between IP3Rs and RyRs into the 17 Å resolution cryo-EM density map of IP3R1 (ref. 34) shows a high correlation coefficient of 0.90. c, Positions of the individual domains in RyR1 (grey) compared to their positions in IP3R1 (coloured). The colour coding is the same as in Fig. 1. Although the middle part of the α-solenoid 1 domain is not conserved, the secondary structure prediction of this region in IP3R1 is similar to RyR1 containing mainly α-helices. We therefore included it in the fit and it agrees well with the density. The complete transmembrane region was included in the fit as the architecture of the ion-channel domain is extremely conserved even between more distant channels. With the exception of RIH2 (2155–2477), which shifted by 70 Å (this domain for RyR1 corresponds to the α-solenoid 2 domain and is omitted from the figure), all other domains had only minor shifts. The large shift of RIH might be caused by the actual shift that occurred as α-solenoid 2 evolved or by reverse directionality of the solenoid domain (Supplementary Information). d, Conserved sequence regions between RyR1 and IP3R1. Numbering corresponds to rabbit RyR1 and human IP3R1, respectively. The most conserved regions are highlighted in bold.

Extended Data Figure 8 Sequence conservation between RyRs and IP3Rs.

a, b, The most conserved regions between RyRs and IP3Rs are in the N-terminal (a, residues 1–432 rabbit RyR1) and C-terminal (b, residues 3,796–5,037 rabbit RyR1) sequence regions. Amino acid sequences were aligned in CLUSTALW2, the amino acids with conservation >50% were coloured in clustalw colour scheme. Domains with the highest conservation are indicated with black bars. Aligned sequences from top to bottom are: rabbit RyR1, human RyR1, rabbit RyR2, human RyR2, rabbit RyR3, human RyR3, Oikopleura dioica RyR, Caenorhabditis elegans RyR, Drosophila melanogaster RyR, Drosophila melanogaster IP3R and human IP3R1, 2 and 3.

Extended Data Figure 9 Conformational dynamics of RyR1 in closed and open states.

ac, Collective motions, changes in the gate region and relative domain movements between different conformations in the closed (a) and open (b, c) states. Left panels: on the right, protomers of the respective two states are shown in cartoon representation, on the left, the conformational changes between them are depicted as vector field. Second panel from left: top view on the pore region depicting the S4–S5 linker and the inner pore helices. Third panel form left: membrane domain, C-terminal domain, α-solenoid 1, and EF-hand of two different conformations in solid-cylinder representation. As in Fig. 3c the structures were aligned to the core of the α-solenoid 1 domain preceding the helices that embed the EF-hand. Similarities between the structures point towards an absence of the specific conformational changes between the conformers from the same data set. Right panels show densities of RyR1 dimers corresponding to different conformations with fitted RyR1 models.

Extended Data Table 1 Structural domains of RyR1

Supplementary information

Supplementary Information

This file contains Supplementary Text, Supplementary Table 1 and additional references. (PDF 456 kb)

Conformational changes between open and closed states

The video shows morphs of cryo-EM maps and structures of RyR1 between closed and open states of the channel. First an overview of conformational changes is shown for a dimer. Then the video zooms on conformational changes at the cytoplasmic surface of the membrane where the ion gate is formed by a bundle of four inner helices (the structure is coloured similar to Figure 1) and conformational changes around EF-hand (purple). To better visualize conformational changes around the EF-hand the density maps and structures of a fragment of a RyR1 protomer were aligned to the α-solenoid 1 region preceding the EF-hand (orange, left from the EF-hand), see also Figure 3. (MOV 20666 kb)

Multiple conformations of RyR1 in closed and open states

Morphs of cryo-EM density maps between two extreme conformations of RyR1 in the closed state and between two conformational transition modes identified in the open state. See also Extended Data Figure 9. (MOV 12085 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Efremov, R., Leitner, A., Aebersold, R. et al. Architecture and conformational switch mechanism of the ryanodine receptor. Nature 517, 39–43 (2015). https://doi.org/10.1038/nature13916

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links