Fig. 5: Tail or 2nd head association has no effect on the ATP-like conformation of the MT-associated Kif5b motor domain.

a Motor domains of the Kif5b^MoNeIAK AMPPNP and MT-bound and ADP-AlF₄ and tubulin-bound (semi-transparent, PDB code 4hna) models were superimposed (left). RMSDs were calculated and displayed on the Kif5b^MoNeIAK model (right), with the Kif5b^MoNeIAK tail shown in magenta. b MT-associated AMPPNP-bound motor domains of the Kif5b^MoNeIAK one (semi-transparent) and two-headed models were superimposed (left). RMSDs were calculated and displayed on the Kif5b^MoNeIAK two-headed model (right) with the Kif5b^MoNeIAK tail shown in magenta and the NL-CC region shown in green. c Overview of the nucleotide-pocket and switch-motif containing loops 9 and 11 (L9 and L11) in the AMPPNP-bound Kif5b^MoNeIAK one-headed model, with side chains for key conserved residues shown. Cryo-EM density is shown as semi-transparent gray and was filtered with DeepEMhancer⁶². d, e Close-up view of residues involved in ATP hydrolysis, comparing d our MT-associated AMPPNP-bound Kif5b^MoNeIAK motor domain (left) and e Eg5 motor domain (PDB code 3hqd). Both models are fitted into density (local resolution sharpened in Relion) for the MT-associated motor domain of AMPPNP-bound Kif5b^MoNeIAK (mesh). The high resolution in the Eg5 motor domain crystal structure allows display of the two water molecules and associated bonding network (black dashed lines) that are involved in nucleophilic attack on the γ-phosphate⁵³. In the right-hand panel, Eg5 residue numbering is shown in black and Kif5b residue numbering in gray. f, g Views of the 2nd (not MT-associated) head in the Kif5b^MoNeIAK two-headed model in the presence of AMPPNP. Cryo-EM density (semi-transparent) was low-pass filtered to 6 Å resolution for resolution-appropriate visualization. The position where density for an extended helix α4 and ordered L11 could be expected (but is absent here) is indicated in semi-transparent colors with dashed black outlines.