Three-dimensional structures of the flagellar dynein-microtubule complex by cryoelectron microscopy

Abstract

The outer dynein arms (ODAs) of the flagellar axoneme generate forces needed for flagellar beating. Elucidation of the mechanisms underlying the chemomechanical energy conversion by the dynein arms and their orchestrated movement in cilia/flagella is of great importance, but the nucleotide-dependent three-dimensional (3D) movement of dynein has not yet been observed. In this study, we establish a new method for reconstructing the 3D structure of the in vitro reconstituted ODA-microtubule complex and visualize nucleotide-dependent conformational changes using cryoelectron microscopy and image analysis. As the complex went from the rigor state to the relaxed state, the head domain of the beta heavy chain shifted by 3.7 nm toward the B tubule and inclined 44 degrees inwards. These observations suggest that there is a mechanism that converts head movement into the axonemal sliding motion.

Figure 1.

SDS-PAGE analysis of the ODA–CB-MT complex. The high-salt extract from Chlamydomonas axonemes (Ex) and the purified ODA–CB-MT complex (Com) were separated by SDS-PAGE and stained with Coomassie brilliant blue. HCs, heavy chains; DC1–3, docking complex protein 1–3; IC1/2, intermediate chain 1/2; LCs, light chains; MW, molecular weight marker.

Figure 2.

EM images of the ODA–CB-MT complex by QFDE, negative-staining, and cryo-EM. A–C, QFDE; D, negative staining; E, cryo-EM. (A) A pair of cross-bridged MTs, as indicated by the arrowheads. The white lines show the row of ODAs. (B) The 24-nm periodicity is indicated by brackets. (C) The globular heads and the thin stalks (arrows) are shown. (D and E) The crossover points are indicated with arrowheads. The black lines indicate the ODAs. (F) The averaged diffraction pattern of the cryo-EM images of five ODA–CB-MT complexes showing the prominent 24-nm layer line. The layer line periodicities are indicated. (G) The interdynein connections observed by cryo-EM. The row of ODAs extending past the end of the MT is indicated with arrowheads. Bars, 100 nm.

Figure 3.

Arrangement of ODAs within the ODA–CB-MT complex and resolution estimation of the reconstructions. (A) A 24-nm filtered cryo-EM image of the ODA–CB-MT complex showing the staggered arrangement of the ODAs. Lines indicate the positions of ODAs. (B) An amplitude and phase plot of the 24-nm layer line from the ODA–CB-MT complex. Phase diff: the phase difference between the near and far sides. The theoretical phase difference is 180° if the two rows of ODAs are staggered. Amplitude: the amplitude of the layer line. The phase value at the amplitude peak (arrowhead) indicates the staggered arrangement of the ODAs. (C) The Fourier shell correlation curves for each reconstruction. The intersection between each curve and the horizontal line at 0.3 was taken as the effective resolution. The effective resolutions are 2.7 nm, 2.7 nm, and 3.5 nm for the rigor state, the relaxed state, and the oda-11 mutant, respectively.

Figure 4.

3D reconstructions of the ODA–CB-MT complex from the wild type in two nucleotide states. A, rigor state; B, relaxed state. Stereo pairs are presented. The color reflects the distance from the center of the MT. The surfaces represent the isosurface of 120% of the volume estimated from the molecular weight of ODA.

Figure 5.

3D reconstructions of the ODA–CB-MT complex in the rigor state and in the relaxed state. A–C, rigor state; D–F, relaxed state. (A and D) The views from the plus end of the MT. (B and E) The side views with the plus end up. (C and F) The 120° rotated side view seen from the inside of the complex. The mesh and solid surfaces represent the isosurface of 120% and 60% volumes, respectively, estimated from the molecular weight of ODA. The domains and fitted objects are colored as follows: the α head is pink, the β head is orange, the γ head is yellow, the base complex is green, and the MTs are light blue. The dark gray isosurface (A and D) shows the position of the protofilaments. The disk-shaped objects were placed according to the center of gravity of the head domain densities, and spherical objects were manually fitted to the densities of the base complex. The atomic models of the tubulins (Lowe et al., 2001) were fitted to the densities of the MT protofilaments (gray). The black arrowheads in B and C indicate the positions of the interdynein connections. The arrowhead in A indicates the position of the connection between the γ head domain and the MT. The red arrowhead in C indicates the connection between the α and β head domains. The orange and yellow arrowheads indicate the base complex β head domain and the base complex γ head domain connections, respectively. In C and F, the β head domain tilts 44° toward the MT in the relaxed state. Bar, 5 nm.

Figure 6.

Demonstration of the motility of the ATP-treated ODA–CB-MT complex. (A) A cryo-EM image of the ATP-treated ODA–CB-MT complex. The ODA–CB-MT complex was treated with 20 μM ATP for 3 min before freezing. The signal of the ODA was enhanced by averaging seven molecules. The overall shape appears triangular. (A and B) The red circles indicate the domains of ODA. (B) A 2D projection of the 3D reconstruction of the ODA–CB-MT complex in the rigor state representing the view in which the ODA is bound to the MT by its base side. The projection resembles the image in A in the position of the domains and the triangular shape. (C) A projection representing the view in which the ODA is bound to the MT by its stalks. (D and E) In vitro motility assay of the ODA–MT complex. (D) The AlexaFluor543-labeled ODA–CB-MT complex was treated with 20 μM ATP and immobilized on the glass coverslips (red). (E) An image sequence of the AlexaFluor488-labeled plain MT (green) showing an MT sliding along the ODA–CB-MT complex. The time interval between each image is 0.5 s. The arrowheads indicate a reference point on the sliding MT. Bar, 5 μm.

Figure 7.

3D reconstruction of the ODA–CB-MT complex from the oda-11 α heavy chain–lacking mutant in the rigor state. (A) A stereo pair of the 3D reconstruction with the plus end up. (B) The view from the plus end of the MT. The arrowhead indicates the expected position of the α heavy chain densities. Bar, 5 nm.

Figure 8.

Cross sections of the ODA–CB-MT complex in the rigor state and in the relaxed state. A–C, rigor state; D–F, relaxed state. The plane of each section is indicated in the top left corner (solid lines). The nucleotide-induced displacement (A and D) and inclination (B and E) of the β head domain is shown. The dotted lines indicate the long axes of the head domains. (A and D) The asterisks indicate the centers of gravity of the β head domain. The gray lines are placed as a reference. The β head domain in the relaxed state displaced 3.7 nm compared with its position in the rigor state. The position of the inter-ODA linker is indicated with arrowheads in C and F. The blue contour corresponds to the 120% volume isosurface; green, yellow, orange, and red indicate increasing density. Bar, 10 nm.

Figure 9.

A comparison of the QFDE-EM images of the axoneme and the corresponding views of the ODA–CB-MT complex in the rigor state and in the relaxed state. (A and C) QFDE-EM images of the axoneme (Goodenough and Heuser, 1982). (B and D) Corresponding views of the ODA–CB-MT complex. Views in the rigor state (A and B) and in the relaxed state (C and D). The densities corresponding to the D foot, P foot, and head are indicated. Bar, 10 nm.

Figure 10.

Head domain displacement between the rigor state and the relaxed state. (A) An end-on view of the 3D reconstruction of the ODA–CB-MT complex in the rigor state. The box used for the slab view in B and C is shown as a dashed rectangle. (B and C) A comparison between the β head domains in the rigor state (B) and the relaxed state (C) showing the change in distance to the B tubule. The double arrows indicate the changes of distance between the β head domain and B tubule from 14 (rigor state) to 10 nm (relaxed state). (D) Model images of the ODA–MT complex in the rigor state (top) and the relaxed state (bottom). The α head domain is omitted from the relaxed state because of the uncertainty of the position. Bar, 5 nm.