This study mapped the conformational landscape of kinesin-1, an essential microtubule motor protein, showing it is highly flexible and favors an open, extended conformation in the absence of cargo. The authors combined single-molecule FRET as their primary technique with Flow Induced Dispersion Analysis (FIDA) to track how ionic strength, mutations and microtubules shift kinesin-1 between open and closed states. FIDA was used to measure the hydrodynamic radius of FLAG-purified kinesin-1 as an independent, ensemble-level confirmation of the smFRET findings. It showed the motor was most extended with no added salt and most compact at 150 mM NaCl, and detected aggregation at high salt. FIDA served as orthogonal, solution-based validation.