The study reveals the molecular mechanism of dynein-dynactin complex assembly by LIS1. Using cryo-electron microscopy, the structure of dynein-dynactin with LIS1 and the lysosomal adaptor JIP3 was determined. This structure shows how JIP3 activates dynein despite its atypical architecture. LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Together, these data suggest LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation. JIP3 is an autoinhibited dynein activating adaptor that requires an intramolecular interaction between its JIP3 helix and the RH1 domain to activate dynein. The structure of dynein-dynactin bound to JIP3 and LIS1 shows that JIP3 residues 24-185 bind along the cleft between the dynein tails and dynactin. The interactions made by the RH1 domain and LZI are sufficient to promote dynein motility. The structure also shows that JIP3 contains a previously unidentified Spindly motif that is critical for the JIP3-pointed end interaction. Dynactin's p150 arm binds DIC-N, DHC and LIS1. The structure raises the question of which interactions are required to open up the CC1A/B hairpin. The data suggest that DIC-N binding opens up CC1A/B to allow CC1B to bind LIS1 and the dynein motor. The IC-LC tower promotes the CC1B/DIC-N interaction. LIS1 stabilizes the pre-powerstroke state of dynein-A. The structure shows that LIS1-N is important for complex assembly. The data suggest that LIS1 binding p150 and dynein is additionally required for DDA complex formation. The study provides a model for the formation of DDA complexes, showing that LIS1 plays roles throughout the initiation of transport and in particular coordinates the interactions required for productive dynein-dynactin assembly. It is thus clear why it is essential for dynein function in cells.The study reveals the molecular mechanism of dynein-dynactin complex assembly by LIS1. Using cryo-electron microscopy, the structure of dynein-dynactin with LIS1 and the lysosomal adaptor JIP3 was determined. This structure shows how JIP3 activates dynein despite its atypical architecture. LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Together, these data suggest LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation. JIP3 is an autoinhibited dynein activating adaptor that requires an intramolecular interaction between its JIP3 helix and the RH1 domain to activate dynein. The structure of dynein-dynactin bound to JIP3 and LIS1 shows that JIP3 residues 24-185 bind along the cleft between the dynein tails and dynactin. The interactions made by the RH1 domain and LZI are sufficient to promote dynein motility. The structure also shows that JIP3 contains a previously unidentified Spindly motif that is critical for the JIP3-pointed end interaction. Dynactin's p150 arm binds DIC-N, DHC and LIS1. The structure raises the question of which interactions are required to open up the CC1A/B hairpin. The data suggest that DIC-N binding opens up CC1A/B to allow CC1B to bind LIS1 and the dynein motor. The IC-LC tower promotes the CC1B/DIC-N interaction. LIS1 stabilizes the pre-powerstroke state of dynein-A. The structure shows that LIS1-N is important for complex assembly. The data suggest that LIS1 binding p150 and dynein is additionally required for DDA complex formation. The study provides a model for the formation of DDA complexes, showing that LIS1 plays roles throughout the initiation of transport and in particular coordinates the interactions required for productive dynein-dynactin assembly. It is thus clear why it is essential for dynein function in cells.