Supplementary Figure 5: Visualization of the native dynein–dynactin–BicD2 complex attached to the microtubule surface.

(a) AMP-PMP, but not ATP, causes dynein to bind MTs. Purified dynein (without added nucleotide) was supplemented with AMP-PNP, ATP or no nucleotide as shown and combined with phosphocellulose-purified, taxol-stabilized MTs. The MTs were pelleted through sucrose, then resusupended in buffer containing 10 mM ATP and pelleted again. The supernatant and pellet from the first centrifugation step (depleted supe and MT pellet) and the final supernatant (ATP release) were analyzed by SDS-PAGE. (b) SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) of dynein-dynactin-BicD2N complex, isolated from mouse brain bound to microtubules. All the components of dynein and dynactin, as well as BicD2N and tubulin are present. (c) Negative stain micrograph showing DDB complexes (indicated by white arrow) attached to the MT surface. Although they are all pointed in the same direction, the DDB complexes are observed at a range of angles relative to the MT surface. (d) Several hundred DDB complexes at a range of angles relative to the MT surface were averaged together to determine the overall dimensions of the complex. The mean distance between the pointed tip of DDB from MT surface is ∼ 33nm, the center of head to the MT surface ∼17nm, and the length of the DDB molecule from the center of head to pointed tip is ∼43nm. (e) Focused classification (see Supplementary figure 2) was used to obtain detailed averages of the motor domains, showing that they are in close proximity to one another when attached to the MT surface. (f) Distribution of angles between DDB and MT surface. The majority of the DDB molecules are oriented between ∼20° and 60° relative to the MT surface. (g) Range of distances between the two heads in isolated dynein and DDB complexes, showing dramatically reduced inter-head distances in the incorporated dynein.