Fig. 2: Kif5b motor domain binding to MTs causes dissociation of the Mg²⁺ ion from ADP and adoption of an apo-like conformation.

a, b Two views of the MT-bound Kif5b^MoNeXXX ADP-state reconstruction with the model fitted into density (gray transparent). In (b), the position where density for a docked neck linker (NL) could be expected (absent here) is indicated in semi-transparent cyan color. c View of the nucleotide pocket of MT-associated Kif5b^MoNeXXX-ADP, highlighting the lack of cryo-EM density at the Mg²⁺ position (semi-transparent lime). d–f Overviews of the nucleotide pocket and switch-motif containing loops 9 (L9) and 11 (L11), with side chains for key mechanistic residues shown for d the crystal structure of Kif5b-ADP motor domain without MTs (PDB code 1bg2) and e our Kif5b^MoNeXXX-ADP structure in the presence of MTs (cryo-EM density in transparent gray). In (f), a superimposition highlighting helix α4’s extension, a twist of the overlying core β-sheet, and concomitant movement of overlying loop 7 (L7) and loop 9 (L9) upon transition from the Kif5b-ADP state without MTs (PDB code 1bg2, semi-transparent model) to our MT-bound Kif5b^MoNeXXX ADP-state (opaque model). Helices are shown as tubes, and αβ-tubulin is shown as a gray surface representation. g, h (c) The crystal structures of the Kif5b motor domain without nucleotide (apo) bound to αβ tubulin dimer (PDB code 4lnu) or (d) of Kif5b-ADP without MTs (PDB code 1bg2) are superimposed on our Kif5b^MoNeXXX ADP-state, and calculated RMSDs are shown. The superimpositions in (f–h) use the nucleotide-holding P-loop and helix α2a elements for alignment. i Kif5b^MoNeXXX ADP-state colored according to the kinesin motor domain subdomain scheme^26,29. Cryo-EM density in all panels was filtered using DeepEMhancer⁶², apart from (c), which was sharpened by local resolution in Relion.