SUMMARY The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of β-tubulin using human Prefoldin and TRiC. We find unstructured β-tubulin is delivered by Prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. CryoEM resolves four near-atomic resolution structures containing progressively folded β-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N-domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Disordered CCT C-termini within the chamber promote subsequent folding of tubulin Core and Middle domains and GTP-binding. Thus, TRiC’s chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

Structural visualization of the tubulin folding pathway directed by eukaryotic chaperonin TRiC