Structural classification of kinesin motor domain using PCA. (A) PCA classified kinesin crystal structures based on their conformational similarity. We projected kinesin structures from the Protein Data Bank—including our crystal and cryo-EM apo structure—onto principal planes defined by the two most significant principal components (PC1 and PC2). Each dot’s color represents one of three classes obtained from hierarchical clustering of the projected structures in the PC1 to PC4 planes. Representative structures of kinesin-1 for each class (1BG2, 1MKJ, 4HNA, 4LNU) and previously reported nucleotide-free structures (1RY6, 3WRD, 5LT1, 5X3E) are labeled with their PDB code. (B) Contribution of each residue (as for human kinesin-1) to the first two principal components (PC1 and PC2). Shaded rectangles indicate the corresponding secondary structures. (C–E) Comparison of representative structures from three classes. 5LT1 was chosen to represent the semi-open state, as the N terminus of its α4 helix extends similar to the microtubule-bound states (cryo-EM apo and 4HNA). These three structures are superposed on the invariant core (gray in C) or the B-domain (gray in D and E). These structures were used to identify subdomains (see Fig. 4).