The K-loop of KIF1A contributes to the clustering of force-generation events. (A) Sequence alignment of KIF1A and KIF5C and sequences of the KIF1A(1–393)-SW and KIF1A(1–393)-SWA mutants. Purple box, loop 12. (B) Number of engagement events per MT encounter (averages during the clustering events per MT encounter are shown in Fig. S3 H). KIF5C(1–560)^C: 1.2 ± 0.1 (mean ± SEM; n = 50); UNC-104(1–389)^E: 2.9 ± 0.3 (n = 92); KIF1A(1–393)^C: 25 ± 4 (n = 50); KIF1A(1–393)-V8M^C: 28 ± 5 (n = 38); KIF1A(1–393)-Y89D^C: 27 ± 4 (n = 31); KIF1A(1–393)^E: 27 ± 3 (n = 50); KIF1A(1–393)-V8M^E: 31 ± 6 (n = 30); KIF1A(1–393)-Y89D^E: 31 ± 5 (n = 30); KIF1A(1–393)-SW^E: 3.6 ± 0.2 (n = 234); KIF1A(1–393)-SWA^E: 3.4 ± 0.2 (n = 286); KIF1A(FL^Act)^C: 58 ± 7 (n = 30); KIF1A(FL^Act)-V8M^C: 59 ± 7 (n = 24); KIF1A(FL^Act)-Y89D^C: 59 ± 8 (n = 20) across two to five independent experiments. ****, P < 0.0001 by unpaired Welch’s t test. (C) Representative force versus time records of bead movement driven by single molecules of KIF1A(1–393)-SW^E (top, k = 0.041 pN/nm) and KIF1A(1–393)-SWA^E (bottom, k = 0.042 pN/nm). (D) Detachment forces. Green bars in B and D indicate the median values with quartiles. KIF1A(1–393)^E: 2.65 (2.25, 3.05) pN, n = 1,044 (from Fig. 1 G and included here for comparison); KIF1A(1–393)-SW^E: 2.00 (1.64, 2.34) pN, n = 1,158; KIF1A(1–393)-SWA^E: 1.84 (1.51, 2.14) pN, n = 1,214 across two to five independent experiments. ****, P < 0.0001 by Welch’s t test. n.s., not significant.