Cryo-EM observation of the proximal end of the neck linker in the nucleotide-free state. (A) 15 Å cryo-EM density map (σ = 1.0) of gold-labeled kinesin–microtubule complex under nucleotide-free condition. A gold cluster was specifically attached to the Cys328 residue on the neck linker. α4 and α6 helices are shown in green and orange, respectively. Red spheres indicate the last ordered residue of the neck linker in the nucleotide-free state (T324). Nucleotide-free kinesin and tubulin crystal structures have been fit to the density map. Extra density corresponding to the attached gold is highlighted in light yellow. The centroid of the gold cluster, identified as the maximum density region (σ = 2.7), is shown as yellow spheres. (B) Structural model of kinesin dimer in the one-head–bound state: the microtubule-bound head is nucleotide-free and in the open state, while the unbound head is in the semi-open state, tethered by disordered neck linkers. The neck linker and the neck coiled-coil are shown in red and blue, respectively.