Figure 1. 3-D helical reconstruction of decorated and undecorated microtubules.

Microtubule/Kar3Cik1 solutions were incubated with apyrase (A) or AMPPNP (B) to generate the nucleotide-free and ATP states respectively. In both states, Kar3Cik1 heterodimers can be seen decorating the microtubules although structural differences between the states cannot be detected. C) 3D helical reconstruction of a plain microtubule. The same result was obtained by incubating Kar3Cik1 motors with ADP prior to addition to microtubules, but where no decoration was detected. A Fourier transform reveals only the 4 nm layer line, which corresponds to the tubulin repeats, illustrating only sparse decoration with motor complexes. D) Reconstruction of a Kar3Cik1 decorated microtubule in the nucleotide-free state. Two distinct densities are visible at each motor subunit corresponding to Kar3 and Cik1. The Fourier transform reveals a strong 4 nm layer line (tubulin repeats) as well as an 8 nm layer line that corresponds to the motor repeat every 8 nm along the microtubule. E) Reconstruction of the ATP state using the non-hydrolyzable ATP analog AMPPNP. The microtubule is fully decorated with Kar3Cik1 as in (D) illustrated by the strong 4 nm and 8 nm layer lines. Corresponding cross-sectional views of the reconstructed microtubules are shown in F) undecorated microtubule, G) nucleotide-free state, and H) AMPPNP state. On the right half of each image, contour lines are shown depicting densities observed from tubulin and Kar3Cik1. Circles corresponding to the position of tubulin (blue), Kar3 (purple), and Cik1 (orange) are shown as well as red circles identifying differences in density between the nucleotide-free and AMPPNP states