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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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 used for the initial separation of singlet and doublet microtubules is available on request from Rui Zhang (firstname.lastname@example.org).
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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.
The authors declare no competing interests.
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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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.
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.
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.
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.
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.
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.
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.
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.
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.
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 Note 1, Supplementary Tables 1−3 and Supplementary Figure 1.
CryoDRGN analysis of RS1 dynamics.
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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