Cryo-EM Reveals the Mechanochemical Cycle of Reactive Full-length Human Dynein-1

Dynein-driven cargo transport plays pivotal roles in diverse cellular activities, central to which is dynein’s mechanochemical cycle. Here, we performed a systematic cryo-electron microscopic investigation of the conformational landscape of full-length human dynein-1 in reaction, under various nucleotide conditions, on and off microtubules. Our approach reveals over 40 high-resolution structures, categorized into eight states, providing a dynamic and comprehensive view of dynein throughout its mechanochemical cycle. The novel intermediate states reveal important mechanistic insights into dynein function, including a ‘backdoor’ phosphate release model that coordinates linker straightening, how microtubule binding enhances ATPase activity through a two-way communication mechanism, and the crosstalk mechanism between AAA1 and the regulatory AAA3 site. Our findings also lead to a substantially revised model for the force-generating powerstroke and reveal a means by which dynein exhibits unidirectional stepping. These results substantially improve our understanding of dynein and provide a more complete model of its mechanochemical cycle.