Fig 2: ?-TuRC assembly starts from a GCP6-stabilized 6-spoke intermediate.a Molecular architecture of the 6-spoke assembly. Numbering of spokes according to the full ?-TuRC and coloring of the components as indicated. Substoichiometry of the ?-TuSC(5,6) and ?-TuSC(13,14) units in this reconstruction is indicated by grey coloring in the schematic representation. b Atomic model (PDB 6X0U) superposed to the segmented density of lumenal bridge module 1. The lumenal bridge components are colored as indicated. Zoom as indicated in panel (a). c Zoom on the MZT1-GCP5 module. Atomic model of the MZT1-GCP5 module (PDB 6L81) superposed to the a-helical bundle on the outer surface of the 6-spoke assembly. d The MZT1-GCP5 module binds to the outer surface of GCP3(8), while MZT1-GCP3(8) remains available to form the first module of the lumenal bridge. e Atomic model of the ?-TuRC including newly built segments of the GCP5 and GCP6 N-terminal regions. Zoom focusing on the interaction of the GCP6-NTE with both copies of GCP4, GCP5, and the lumenal bridge components. Interactions are indicated with asterisks. Coloring as in (a).

Fig 3: Mechanistic model for modular assembly of the human ?-TuRC.a The initial step of ?-TuRC assembly is the formation of the 6-spoked stable core consisting of GCP2-3-4-5-4-6, in which the MZT1-GCP3 module is stabilized via the GCP6 GRIP1-NTE. Coloring as indicated. b Assembly continues (yellow arrow) via successive addition of ?-TuSC units. In this expansion phase, ?-TuSC units are conformationally locked (schematic lock symbol) when neighboring ?-TuSC units associate to the assembly intermediates (lighter color). During this process, the ?-TuSC(13,14) unit can repeatedly associate and dissociate. It is repositioned as soon as the lumenal bridge has formed. c Expansion ends (green arrow) in-ring closure, where the ?-TuSC(1,2) unit is integrated, stabilizes terminally the ?-TuSC(13,14) unit and locks the fully assembled 14-spoked complex.

Fig 4: Actin is not required for ?-TuRC assembly but impacts on ?-TuRC geometry.a The very N-terminus of GCP6 is crucial for actin binding. Immunoblot analysis of an actin IP experiment against His tagged, N-terminal fragments of GCP6 (GCP6N126-His8; GCP6N57-126-His8), co-expressed with FLAG-MZT1 in E. coli. Input and IP samples were probed against anti-actin, anti-His (GCP6) and anti-FLAG (MZT1) antibodies. Sections of representative immunoblots of one experiment are shown. IP experiments were performed in (n = 3) independent experiments and source data are provided as a Source Data file. b Cryo-EM reconstruction of ?-TuRC?N56-GCP6 deficient in actin binding. Coloring as indicated. Spokes are numbered. c Structure and composition of the lumenal bridge in ?-TuRC?N56-GCP6. Atomic model of resolved components (PDB 6X0U) superposed to the segmented density of the lumenal bridge. The structure is devoid of cryo-EM density for the two N-terminal helices of GCP6 and actin. Coloring as indicated. d Effect of actin integration on ?-TuRC geometry, as represented by vectors linking the Ca atoms in both conformations. Vectors are colored according to scale bar. Directionality of motion is represented by an arrow.