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Structures of radial spokes and associated complexes important for ciliary motility

Abstract

In motile cilia, a mechanoregulatory network is responsible for converting the action of thousands of dynein motors bound to doublet microtubules into a single propulsive waveform. Here, we use two complementary cryo-EM strategies to determine structures of the major mechanoregulators that bind ciliary doublet microtubules in Chlamydomonas reinhardtii. We determine structures of isolated radial spoke RS1 and the microtubule-bound RS1, RS2 and the nexin−dynein regulatory complex (N-DRC). From these structures, we identify and build atomic models for 30 proteins, including 23 radial-spoke subunits. We reveal how mechanoregulatory complexes dock to doublet microtubules with regular 96-nm periodicity and communicate with one another. Additionally, we observe a direct and dynamically coupled association between RS2 and the dynein motor inner dynein arm subform c (IDAc), providing a molecular basis for the control of motor activity by mechanical signals. These structures advance our understanding of the role of mechanoregulation in defining the ciliary waveform.

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Fig. 1: Structures of radial spokes on and off doublet microtubules.
Fig. 2: Structural basis for the microtubule docking and longitudinal periodicity of radial spokes.
Fig. 3: Structure of the radial spoke head.
Fig. 4: Interactions between RSP3 and radial spoke proteins.
Fig. 5: IDA subforms a and c dock onto the bases of radial spokes.
Fig. 6: Molecular basis for the control of IDA motor activity by mechanical signals.

Data availability

Composite cryo-EM maps and atomic models have been deposited in the Electron Microscopy Data Bank (EMDB) and wwPDB, respectively, under accession codes EMD-22475 and PDB 7JTK (isolated RS1) and EMD-22481 and PDB 7JU4 (on-doublet RS2 stalk/IDAc/N-DRC). Constituent maps and the masks that were applied during reconstruction are associated with these depositions as additional files. Cryo-EM maps have been deposited under accession codes EMD-22480 (on-doublet RS1 stalk), with associated atomic model PDB 7JTS, EMD-22482 (on-doublet RS1 spoke head), EMD-22483 (on-doublet RS2 spoke head) and EMD-22486 (on-doublet RSP1 dimer).

Code availability

Code used for the initial separation of singlet and doublet microtubules is available on request from Rui Zhang (zhangrui@wustl.edu).

References

  1. 1.

    Ginger, M. L., Portman, N. & McKean, P. G. Swimming with protists: perception, motility and flagellum assembly. Nat. Rev. Microbiol. 6, 838–850 (2008).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. 2.

    Bustamante-Marin, X. M. & Ostrowski, L. E. Cilia and mucociliary clearance. Cold Spring Harb. Perspect. Biol. 9, a028241 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  3. 3.

    Lyons, R. A., Saridogan, E. & Djahanbakhch, O. The reproductive significance of human fallopian tube cilia. Hum. Reprod. Update 12, 363–372 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. 4.

    Faubel, R., Westendorf, C., Bodenschatz, E. & Eichele, G. Cilia-based flow network in the brain ventricles. Science 353, 176–178 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  5. 5.

    Nonaka, S. et al. Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell 95, 829–837 (1998).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    Ishikawa, T. Axoneme structure from motile cilia. Cold Spring Harb. Perspect. Biol. 9, a028076 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  7. 7.

    Brokaw, C. J. & Kamiya, R. Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function. Cell Motil. Cytoskeleton 8, 68–75 (1987).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. 8.

    Lin, J. & Nicastro, D. Asymmetric distribution and spatial switching of dynein activity generates ciliary motility. Science 360, eaar1968 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  9. 9.

    Heuser, T., Raytchev, M., Krell, J., Porter, M. E. & Nicastro, D. The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella. J. Cell Biol. 187, 921–933 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Bower, R. et al. The N-DRC forms a conserved biochemical complex that maintains outer doublet alignment and limits microtubule sliding in motile axonemes. Mol. Biol. Cell 24, 1134–1152 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Luck, D., Piperno, G., Ramanis, Z. & Huang, B. Flagellar mutants of Chlamydomonas: studies of radial spoke-defective strains by dikaryon and revertant analysis. Proc. Natl Acad. Sci. USA 74, 3456–3460 (1977).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12.

    Smith, E. F. & Sale, W. S. Regulation of dynein-driven microtubule sliding by the radial spokes in flagella. Science 257, 1557–1559 (1992).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  13. 13.

    Pigino, G. et al. Cryoelectron tomography of radial spokes in cilia and flagella. J. Cell Biol. 195, 673–687 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Barber, C. F., Heuser, T., Carbajal González, B. I., Botchkarev, V. V. & Nicastro, D. Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella. Mol. Biol. Cell 23, 111–120 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Lin, J. et al. Cryo-electron tomography reveals ciliary defects underlying human RSPH1 primary ciliary dyskinesia. Nat. Commun. 5, 5727 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Lin, J. et al. Building blocks of the nexin-dynein regulatory complex in Chlamydomonas flagella. J. Biol. Chem. 286, 29175–29191 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Piperno, G., Huang, B. & Luck, D. J. Two-dimensional analysis of flagellar proteins from wild-type and paralyzed mutants of Chlamydomonas reinhardtii. Proc. Natl Acad. Sci. USA 74, 1600–1604 (1977).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18.

    Piperno, G., Huang, B., Ramanis, Z. & Luck, D. J. Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components. J. Cell Biol. 88, 73–79 (1981).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  19. 19.

    Yang, P., Diener, D. R., Rosenbaum, J. L. & Sale, W. S. Localization of calmodulin and dynein light chain LC8 in flagellar radial spokes. J. Cell Biol. 153, 1315–1326 (2001).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Reiter, J. F. & Leroux, M. R. Genes and molecular pathways underpinning ciliopathies. Nat. Rev. Mol. Cell Biol. 18, 533–547 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Ma, M. et al. Structure of the decorated ciliary doublet microtubule. Cell 179, 909–922.e12 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Oda, T., Yanagisawa, H., Kamiya, R. & Kikkawa, M. Cilia and flagella. A molecular ruler determines the repeat length in eukaryotic cilia and flagella. Science 346, 857–860 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    Lin, H. et al. A NIMA-related kinase suppresses the flagellar instability associated with the loss of multiple axonemal structures. PLoS Genet. 11, e1005508 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  24. 24.

    Dymek, E. E., Heuser, T., Nicastro, D. & Smith, E. F. The CSC is required for complete radial spoke assembly and wild-type ciliary motility. Mol. Biol. Cell 22, 2520–2531 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Urbanska, P. et al. The CSC proteins FAP61 and FAP251 build the basal substructures of radial spoke 3 in cilia. Mol. Biol. Cell 26, 1463–1475 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Oda, T., Yagi, T., Yanagisawa, H. & Kikkawa, M. Identification of the outer-inner dynein linker as a hub controller for axonemal dynein activities. Curr. Biol. 23, 656–664 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27.

    Poghosyan, E. et al. The structure and symmetry of radial spoke protein complex in Chlamydomonas flagella. J. Cell. Sci. 133, jcs.245233 (2020).

    Article  CAS  Google Scholar 

  28. 28.

    Yang, C., Compton, M. M. & Yang, P. Dimeric novel HSP40 is incorporated into the radial spoke complex during the assembly process in flagella. Mol. Biol. Cell 16, 637–648 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29.

    Zhu, X. et al. The roles of a flagellar HSP40 ensuring rhythmic beating. Mol. Biol. Cell 30, 228–241 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    El Khouri, E. et al. Mutations in DNAJB13, encoding an HSP40 family member, cause primary ciliary dyskinesia and male infertility. Am. J. Hum. Genet. 99, 489–500 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Warner, F. D. & Satir, P. The structural basis of ciliary bend formation. Radial spoke positional changes accompanying microtubule sliding. J. Cell Biol. 63, 35–63 (1974).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Goodenough, U. W. & Heuser, J. E. Substructure of inner dynein arms, radial spokes, and the central pair/projection complex of cilia and flagella. J. Cell Biol. 10, 2008–2018 (1985).

    Article  Google Scholar 

  33. 33.

    Kubo, T., Hou, Y., Cochran, D. A., Witman, G. B. & Oda, T. A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1). Mol. Biol. Cell 29, 1060–1074 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Qin, H., Diener, D. R., Geimer, S., Cole, D. G. & Rosenbaum, J. L. Intraflagellar transport (IFT) cargo IFT transports flagellar precursors to the tip and turnover products to the cell body. J. Cell Biol. 164, 255–266 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Diener, D. R. et al. Sequential assembly of flagellar radial spokes. Cytoskeleton (Hoboken) 68, 389–400 (2011).

    CAS  Article  Google Scholar 

  36. 36.

    Gupta, A., Diener, D. R., Sivadas, P., Rosenbaum, J. L. & Yang, P. The versatile molecular complex component LC8 promotes several distinct steps of flagellar assembly. J. Cell Biol. 198, 115–126 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Oda, T., Yanagisawa, H., Yagi, T. & Kikkawa, M. Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity. J. Cell Biol. 204, 807–819 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Gaillard, A. R., Diener, D. R., Rosenbaum, J. L. & Sale, W. S. Flagellar radial spoke protein 3 is an A-kinase anchoring protein (AKAP). J. Cell Biol. 153, 443–448 (2001).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39.

    LeDizet, M. & Piperno, G. The light chain p28 associates with a subset of inner dynein arm heavy chains in Chlamydomonas axonemes. Mol. Biol. Cell 6, 697–711 (1995).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Kagami, O. & Kamiya, R. Translocation and rotation of microtubules caused by multiple species of Chlamydomonas inner-arm dynein. J. Cell. Sci. 103, 653–664 (1992).

    CAS  Google Scholar 

  41. 41.

    Yanagisawa, H.-A. & Kamiya, R. Association between actin and light chains in Chlamydomonas flagellar inner-arm dyneins. Biochem. Biophys. Res. Commun. 288, 443–447 (2001).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Houdusse, A. et al. Crystal structure of apo-calmodulin bound to the first two IQ motifs of myosin V reveals essential recognition features. Proc. Natl Acad. Sci. USA 103, 19326–19331 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. 43.

    Shen, M. et al. Calmodulin in complex with the first IQ motif of myosin-5a functions as an intact calcium sensor. Proc. Natl Acad. Sci. USA 113, E5812–E5820 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  44. 44.

    Bessen, M., Fay, R. B. & Witman, G. B. Calcium control of waveform in isolated flagellar axonemes of Chlamydomonas. J. Cell Biol. 86, 446–455 (1980).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    King, S. M. & Patel-King, R. S. Identification of a Ca2+-binding light chain within Chlamydomonas outer arm dynein. J. Cell. Sci. 108, 3757–3764 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Price, M. E. & Sisson, J. H. Redox regulation of motile cilia in airway disease. Redox Biol. 27, 101146 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Lindemann, C. B. A cAMP-induced increase in the motility of demembranated bull sperm models. Cell 13, 9–18 (1978).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  48. 48.

    Smith, E. F. & Yang, P. The radial spokes and central apparatus: mechano-chemical transducers that regulate flagellar motility. Cell Motil. Cytoskeleton 57, 8–17 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49.

    Yang, P. et al. Radial spoke proteins of Chlamydomonas flagella. J. Cell. Sci. 119, 1165–1174 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Lindemann, C. B. & Lesich, K. A. The geometric clutch at 20: stripping gears or gaining traction? Reproduction 150, R45–R53 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  51. 51.

    Yagi, T. et al. An axonemal dynein particularly important for flagellar movement at high viscosity. Implications from a new Chlamydomonas mutant deficient in the dynein heavy chain gene DHC9. J. Biol. Chem. 280, 41412–41420 (2005).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  52. 52.

    Kamiya, R. Extrusion and rotation of the central-pair microtubules in detergent-treated Chlamydomonas flagella. Prog. Clin. Biol. Res. 80, 169–173 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Omoto, C. K. et al. Rotation of the central pair microtubules in eukaryotic flagella. Mol. Biol. Cell 10, 1–4 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. 54.

    Mitchell, D. R. Orientation of the central pair complex during flagellar bend formation in Chlamydomonas. Cell Motil. Cytoskeleton 56, 120–129 (2003).

    PubMed  Article  PubMed Central  Google Scholar 

  55. 55.

    Satouh, Y. & Inaba, K. Proteomic characterization of sperm radial spokes identifies a novel spoke protein with an ubiquitin domain. FEBS Lett. 583, 2201–2207 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  56. 56.

    Sager, R. & Granick, S. Nutritional control of sexuality in Chlamydomonas reinhardi. J. Gen. Physiol. 37, 729–742 (1954).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. 57.

    Craige, B., Brown, J. M. & Witman, G. B. Isolation of Chlamydomonas flagella. Curr. Protoc. Cell Biol. 59, 3.41.1–3.41.9 (2013).

    Article  Google Scholar 

  58. 58.

    Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, 163 (2018).

    Article  Google Scholar 

  59. 59.

    Schorb, M., Haberbosch, I., Hagen, W. J. H., Schwab, Y. & Mastronarde, D. N. Software tools for automated transmission electron microscopy. Nat. Methods 16, 471–477 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  60. 60.

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

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  61. 61.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. 62.

    Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    PubMed  PubMed Central  Article  Google Scholar 

  63. 63.

    Wagner, T. et al. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2, 218–13 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  64. 64.

    Zhang, K. et al. Cryo-EM reveals how human cytoplasmic dynein is auto-inhibited and activated. Cell 169, 1303–1314.e18 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  65. 65.

    Nakane, T., Kimanius, D., Lindahl, E. & Scheres, S. H. Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION. Elife 7, 1485 (2018).

    Article  Google Scholar 

  66. 66.

    Scheres, S. H. W. In Methods in Enzymology Vol 579 (Ed. Crowther, R. A.) 125–157 (Elsevier, 2016).

  67. 67.

    Zhong, E. D., Bepler, T., Berger, B. & Davis, J. H. CryoDRGN: reconstruction of heterogeneous structures from cryo-electron micrographs using neural networks. Preprint at bioRxiv https://doi.org/10.1101/2020.03.27.003871 (2020).

  68. 68.

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  69. 69.

    Pazour, G. J., Agrin, N., Leszyk, J. & Witman, G. B. Proteomic analysis of a eukaryotic cilium. J. Cell Biol. 170, 103–113 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  70. 70.

    Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D Struct. Biol. 74, 531–544 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  71. 71.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  Article  Google Scholar 

  72. 72.

    Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    CAS  PubMed  Article  Google Scholar 

  73. 73.

    Morin, A. et al. Collaboration gets the most out of software. Elife 2, e01456 (2013).

    PubMed  PubMed Central  Article  Google Scholar 

  74. 74.

    Lechtreck, K. F., Mengoni, I., Okivie, B. & Hilderhoff, K. B. In vivo analyses of radial spoke transport, assembly, repair and maintenance. Cytoskeleton (Hoboken) 75, 352–362 (2018).

    CAS  Article  Google Scholar 

  75. 75.

    Scoble, J. et al. Crystal structure and comparative functional analyses of a Mycobacterium aldo-keto reductase. J. Mol. Biol. 398, 26–39 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  76. 76.

    Hirano, Y., Kimura, S. & Tamada, T. High-resolution crystal structures of the solubilized domain of porcine cytochrome b5. Acta Crystallogr. D Biol. Crystallogr. 71, 1572–1581 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  77. 77.

    Gamble, T. R. et al. Crystal structure of human cyclophilin A bound to the amino-terminal domain of HIV-1 capsid. Cell 87, 1285–1294 (1996).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  78. 78.

    Martinez, S. E. et al. Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: modes of ligand binding and dimerization. Proc. Natl Acad. Sci. USA 102, 3082–3087 (2005).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

Download references

Acknowledgements

Cryo-EM data were collected at the Washington University in St. Louis Center for Cellular Imaging (WUCCI) and the Harvard Cryo-Electron Microscopy Center for Structural Biology. We thank J. Fitzpatrick, M. Rau, S. Sterling and R. Walsh for microscopy support; R. Tomaino for MS analysis; and T. Walton for comments. M.G. is supported by a BCMP-Merck postdoctoral fellowship. B.B. is supported by NIH grant R01-GM081871. J.H.D. is supported by NIH grant R00-AG050749. B.B. and J.H.D. are supported by the MIT J-Clinic for Machine Learning and Health. S.K.D. is supported by NIGMS grant R35-GM131909. Research in the Brown laboratory is supported by the E. Matilda Ziegler Foundation for the Blind, the Smith Family Foundation and the Pew Charitable Trusts.

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Contributions

M.G., M.M., E.S.-T. and F.K. prepared samples; M.G., M.M. X.W. and R.Z. collected and processed cryo-EM data; M.G. and A.B. built the atomic models; E.D.Z., B.B. and J.H.D. developed cryoDRGN and performed dynamics analysis; and M.G. and R.Z. performed multibody analysis. S.K.D., R.Z. and A.B. supervised the research. R.Z. and A.B. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Rui Zhang or Alan Brown.

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The authors declare no competing interests.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Data collection and processing for the on-doublet complexes.

a, Section of an electron micrograph showing radial spokes (marked with an asterisk) bound to a doublet microtubule in vitreous ice. b, Processing scheme used to generate reconstructions of complexes bound to doublet microtubules (DMT, gray). To resolve various structural features with 96-nm periodicity (RS1 spoke head/stalk, RS2 spoke head/stalk, RSP1-RSP1 interface, RS3S, IDAc, or N-DRC baseplate/lobe), it was necessary to use a combination of tubulin signal subtraction (TSS), shifting the center (SC) of coordinates to the feature of interest, focused refinement (FR) and 3D classification without alignment (3C). When possible, the box size was reduced (RB) to 256 or 384 instead of 512 pixels to facilitate data processing. c, Angular distribution of the particle views used for reconstruction of on-doublet RS2. Similar distributions were obtained for on-doublet RS1. The height of the cylinders, colored from blue to red, represents the number of particles. The final density map of RS2 is shown in gray. d, Superimposition of the on-doublet RS1 and RS2 spoke heads confirms that they have identical structure.

Extended Data Fig. 2 Global and local resolution of on-doublet complexes.

a, Fourier shell correlation (FSC) curves calculated between masked independent half maps for on-doublet structures. Left panel, FSC curves are shown for focused refinements of the RS1 spoke head and stalk, RS2 spoke head and stalk, and the RSP1-RSP1 dimer. Right panel, FSC curves for the base of RS1, the RS2-IDAc complex, IDAc, and the N-DRC baseplate. The nominal resolution was estimated using the FSC = 0.143 criterion (dashed line). b, Density maps for on-doublet structures colored by local resolution. Only the maps used for model building are shown. The local resolution is colored from 3 to 7 Å.

Extended Data Fig. 3 Map quality.

Examples of map density for all 30 non-tubulin proteins identified in this study. The first 19 proteins show density from isolated RS1 contoured at 0.009-0.013. The remaining 11 proteins (starting from RSP8) show density from on-doublet maps contoured at 0.020-0.031. Landmark residues are labeled. Note that the sidechains of RSP20 (calmodulin) and RSP8 are not well resolved and are truncated in the deposited model.

Extended Data Fig. 4 Single-particle cryo-EM maps docked into a subtomogram average of the axoneme.

a, Two views showing the single-particle cryo-EM maps of RS1, RS2, RS3S, N-DRC, and IDAc docked into the subtomogram average of the 96-nm repeat of the Chlamydomonas axoneme (EMD-6872). The subtomogram average is shown as a gray isosurface. b, Zoom-in view showing the map of RS3S. Density for RS3S is recovered in 25% of the particles following 3D classification (Extended Data Fig. 1b). RS3S interacts with two molecular staples of unknown identity. c, Zoom-in view showing the model and map for the N-DRC baseplate. Three N-DRC subunits (DRC1, DRC2, and DRC4) can be unambiguously identified. FAP91 interacts with all three.

Extended Data Fig. 5 Data collection and processing for isolated RS1.

a, Chromatogram showing the elution of RS1 from an anion-exchange column using a KCl gradient. The peak fraction containing RS1 is highlighted and elutes at ~0.7 M KCl. b, Silver-stained SDS-PAGE gel showing the purity of isolated RS1 following anion-exchange chromatography. The molecular weights of markers (in kDa) are indicated on the left. The result of mass spectrometry analysis of this sample is given in Supplementary Data 1. c, Section of a negative-stain electron micrograph showing homogeneous and monodisperse radial spokes. d, Selected two-dimensional class averages of particles selected from negative-stain electron micrographs. e, Section of an electron micrograph showing radial spokes in vitreous ice. Particles showing the characteristic T-shaped projection of radial spokes are circled. f, Selected two-dimensional class averages of radial spokes showing well defined spoke heads but nebulous density for the stalk consistent with flexibility at the neck. g, Schematic showing the processing of the isolated RS1 data. Following a consensus refinement, the spoke head and stalk were independently refined. The twofold rotational symmetry of the spoke head was exploited to improve the map quality. Further masked refinement was used to improve the flexible projections of the spoke head and the base and neck of the stalk. These individual maps were recombined to generate a final composite cryo-EM map. h, Angular distribution of the particle views used for the consensus reconstruction of isolated RS1. The height of the cylinders, colored from blue to red, represents the number of particles. The final density map of RS1 is shown in gray.

Extended Data Fig. 6 Global and local resolution of isolated RS1.

a, FSC curves calculated between masked independent half maps for isolated RS1. Left panel, FSC curves are shown for the consensus refinement of isolated RS1, focused refinement of the stalk, and focused refinement of the spoke head after applying C2 symmetry. Right panel, FSC curves for focused refinements of three subdomains of a single lobe of the RS1 spoke head. The colors of the curves match the masks used in Extended Data Fig. 5g. The nominal resolution was estimated using the FSC=0.143 criterion (dashed line). b, Density maps for the consensus refinement of isolated RS1 and various focused refinements colored by local resolution. The local resolution is colored from 3 to 7 Å.

Extended Data Fig. 7 The stalks of RS1 and RS2.

a, The stalk of the isolated radial spoke is consistent with the on-doublet stalk of RS1 only. FAP253, RSP14, and calmodulin are present in the stalk of RS1 but not RS2. RSP8, RSP15, and an unidentified ubiquitin (Ub)-like domain are present in the stalk of RS2 but not RS1. LC8, FAP207, and RSP3 are common to both RS1 and RS2 but adopt different conformations. The RSP7/11 heterodimer is similar in both radial spokes. b, RSP14 and RSP8 are structurally similar armadillo proteins present in different radial spokes. Left, RSP14 was identified in the stalk of RS1 based on well-defined sidechain density. Middle, the model of RSP14 is incompatible with the density of the armadillo protein in RS2, indicating that they are different proteins with similar folds. Right, a model for RSP8 built into the RS2 density. c, Superposition of the atomic models for RSP8 and RSP14.

Extended Data Fig. 8 Model of radial spoke assembly.

Proposed model of radial spoke assembly. Monomeric spoke head lobes, comprising RSP1-7 and RSP9-12, assemble in the cell body28,34,35,36 before being imported into the cilium by intraflagellar transport (IFT)34,74. In the cilium, the axonemal doublet microtubules are bound by the CCDC39–40 coiled coil. Specific sequences within the coiled coil are recognized by molecular adaptors FAP253 and FAP91 that establish the binding sites for RS1 and RS2. These molecular adaptors recruit LC8 and FAP207, although the arrangement of these elements is different in the two stalks. RSP3 in the precursor binds the LC8 multimers (Fig. 4b), helping dock the spoke head lobe onto the preassembled stalks. Two lobes can bind a single stalk. Binding of RSP16 is presumably a relatively late step that dimerizes the lobes28. At a similar time, RS-specific proteins bind; RSP14 to RS1 and RSP8 to RS2.

Extended Data Fig. 9 Dynamics of radial spokes by multi-body analysis.

a, Multi-body analysis of isolated RS1. Left, the contributions of all eigenvectors to the variance. The first eigenvector accounts for 37% of all variability. Inset, the unimodal histogram of amplitudes along the first eigenvector indicates continuous motion. Right, the density maps at the extremes and middle show the same tilting of the spoke head relative to the stalk as observed by the neural-network approach in Fig. 6a. b, Multi-body analysis of on-doublet RS1 shows the same direction of spoke head tilt as isolated RS1. c, Multi-body analysis of on-doublet RS2 shows that the spoke heads of both radial spokes tilt in similar directions to similar extents. d, Multi-body analysis of the movement of the RS1 stalk with respect to the doublet microtubule (DMT) surface.

Extended Data Fig. 10 Potential chemical modulation of radial spokes.

a, Calmodulin binds the IQ motif of FAP253 at the base of RS1. Below, sequence of FAP253 residues 400-430 showing the presence of an IQ motif (emboldened with motif-defining residues boxed). b, Structural comparison of calmodulin bound to FAP253 with apo-calmodulin bound to an IQ motif from myosin V (PDB 2IX7)42. c, Structural comparison of calmodulin bound to FAP253 with Ca2+-calmodulin bound to an IQ motif from myosin 5a (PDB 4ZLK)42. d, The structure of RSP5 resembles an NADPH-dependent aldo-keto reductase domain (PDB 2WZM)75. However, the NADPH binding site of RSP5 is absent and filled by two loops (residues 393-414 and 468-484 of RSP5). e, The N-terminal domain of FAP198 closely resembles heme-binding cytochrome b5 (PDB 3X34)76. However, no heme is observed bound to FAP198, and the putative heme-binding site is occluded by a loop of FAP198 (residues 89–95). f, RSP12 structurally resembles cylophilin-type peptidyl-prolyl cis-trans isomerase (PDB 1AK4)77. The putative substrate-binding site of RSP12 is occupied by a loop of FAP198 (residues 96–105), which positions a proline (P99) in the active site. g, Atomic model of the GAF domain from RSP2 superposed with the model of a cAMP-bound GAF domain (PDB 1YKD)78. Unexplained density in the RSP2 GAF domain (pink, contoured at 0.01) is observed in the cAMP binding pocket, but the resolution is insufficient to assign it as a cyclic nucleotide. The cAMP ligand from PDB 1YKD is shown for comparison. h, Atomic model of RSP23 superposed with an active, ADP-bound nucleoside diphosphate kinase (NDK; PDB 4HR2). Many of the active site residues are conserved. Potential density for a bound nucleotide to RSP23 is observed in the on-doublet map of RS1 (purple, contoured at 0.017) but not in the isolated RS1 map. The ADP ligand from PDB 4HR2 is shown for comparison.

Supplementary information

Supplementary Information

Supplementary Note 1, Supplementary Tables 1−3 and Supplementary Figure 1.

Reporting Summary

Supplementary Video 1

CryoDRGN analysis of RS1 dynamics.

Supplementary Data 1

Mass spectrometry analysis of purified RS1. Proteins identified in the cryo-EM density maps are highlighted in yellow.

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Gui, M., Ma, M., Sze-Tu, E. et al. Structures of radial spokes and associated complexes important for ciliary motility. Nat Struct Mol Biol 28, 29–37 (2021). https://doi.org/10.1038/s41594-020-00530-0

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