Crystal structures of G234A mutant kinesin-1 motor domain. (A) 2.4 Å crystal structure of Cys-light mutant kinesin-1 (336 amino acids) with G234A mutation complexed with ADP (yellow), aligned with the ADP-bound structure of wild-type human kinesin-1 (blue; PDB# 1BG2 [Kull et al., 1996]). The root mean square deviation (RMSD) of backbone atoms between these structures was 0.819 Å. The G234A residue is highlighted as a green sphere, and cysteine substitutions are shown as red spheres. (B) 2.8 Å crystal structure of nucleotide-free G234A mutant kinesin-1 motor domain (yellow), aligned with the nucleotide-free motor domain from the kinesin–tubulin complex crystal structure (blue; #4LNU [Cao et al., 2014]). The RMSD of backbone atoms between these structures was 1.259 Å. L11 is disordered in our crystal structure but ordered in 4LNU, suggesting that microtubule binding is necessary for L11 folding. (C and D) Close-up views of the α4 and α6 helices in nucleotide-free and ADP·AlF₄-bound (green; #4HNA [Gigant et al., 2013]) states. The initial residues of the neck linker (K323, T324, and I325) are highlighted as ball-and-stick models.