Helical 3D reconstruction of dynein-microtubule complex. (A) Cryo-electron micrograph of a microtubule highly decorated with recombinant dynein molecules. Scale bar, 1,000 Å. (B) Helically averaged image of the dynein-microtubule complex (blue chicken wire). The microtubule is shown at a different threshold level (gray contour). (Scale bar, 100 Å.)

Single particle 3D reconstruction of dynein-microtubule complex. (A) Cryo-electron micrograph of a microtubule sparsely decorated with recombinant dynein molecules. (Scale bar, 1,000 Å.) Phases are flipped to be positive in this figure. The red arrowheads indicate dynein molecules. (B) Representative class averages of the dynein-microtubule complex. (C) 3D reconstruction of the dynein (H380)-microtubule complex. The resolution was cut off at 25 Å. The microtubule plus end is facing up in the top view. (D) Back and front views of the dynein head domain. The red arrowhead indicates the density spanning the central hole in the AAA+ ring.

Three-dimensional reconstruction of dynein stalk–microtubule complex. (A and B) Helical reconstructions of a microtubule decorated with DS viewed from the plus end of the microtubule (A) and from the side, with the plus end facing up (B). (C) The dynein-microtubule reconstruction with a stalk prediction (blue blurred line). (D–G) Projection maps of the dynein-microtubule complex (D and E) and the DS-microtubule complex (F and G). The outline of the DS-microtubule complex (pink) is overlaid over that of the dynein-microtubule complex (black). The blue and gray lines in D indicate the directions in which the stalk could extend from the DS-microtubule reconstruction. The blue line connects to the dynein head and is the likely position of the stalk.

Schematic diagrams of the dynein constructs. (A) Proposed domain structure of the dynein heavy chain. The dashed line represents the region that was deleted from the expression construct. (B) Domain organization of the dynein heavy chain. Numbers 1–6 indicate the six AAA+ domains. The two bars between AAA4 and AAA5 represent helices that form an antiparallel coiled coil. The structurally undefined C terminus seventh domain was indicated with “C.”

Orientation of dynein head bound to microtubules. (A and B) Two possible types of dynein flexibility. (A) Rotation of the AAA+ ring about the axis of the stalk. (B) Bending of the AAA+ ring. (C) Distributions of dynein particles showing the face view (O, red), oblique view (0, blue), or side view (I, yellow) of the AAA+ ring. The ratio was normalized to make the total 1 for each distance from the microtubule center. The black line in the graph indicates the edge of the microtubule. (D) Projection averages of dynein particles at a given distance from the center of the microtubule.

Locating subdomains of dynein head. (A) The H380 reconstruction shows seven distinct densities. (B) Projected density of the head domain seen in the same orientation as the 3D density map in A. Numbers 1–7 label densities representing individual AAA+ domains. (C–E) 2D averages of the H380 (C), H-GFP-380 (D), and H-GFP-380-B2 (E) constructs. Dyneins within the 25.2° arc were averaged, causing the Moiré patterns of the microtubule to be smeared out. (F–I) Student's t test results between the averages shown in C and D with P = 0.004 (green) (F), C and E with P = 0.05 (blue) (G), D and E with P = 0.004 (red) (H), and the merged map of F–H (I), respectively. P is the probability that the difference between two datasets is insignificant.

Models showing how a conformational change in the dynein domain could generate movement along a microtubule. The N terminus of the dynein heavy chain, which is not included in the construct, is shown in dotted line. Our construct starts from the GFP tag (green), followed by the linker (yellow) and AAA+ domains, which include BFP at AAA2 (blue). The GFP tag is located on the periphery of the AAA+ ring that faces the plus end of the microtubule. The numbering in the AAA+ ring was done based on our research results. The angle between the tail and the stalk is drawn to be consistent with a previous study (7). The movement of the tail during the transition from states II to I would result in a translocation of dynein toward the minus end of the microtubule. (A) In state II, in which dynein is not tightly bound to the microtubule, the tail is closer to AAA2 than to the C terminus. The arrangement of the AAA+ domains is clockwise (arrow, state II) in AAA+ ring. The stalk is tilted toward the microtubule in state II, and perpendicular to it in state I, so that the power stroke results in a movement of the dynein head toward the minus end of the microtubule. (B) The AAA+ domains are arranged counterclockwise (arrow, state II). The stalk is perpendicular to the microtubule in state II and tilts toward the microtubule in state I, so that the power stroke results in a movement of the dynein head toward the minus end of the microtubule.