4.0 Å Resolution Cryo‐EM Structure of the Mammalian Chaperonin TRiC/CCT Reveals its Unique Subunit Arrangement

Abstract

TRiC is a eukaryotic chaperonin essential for de novo folding of ~10% newly synthesized cytosolic proteins, many of which cannot be folded by other chaperones. This is likely linked to TRiC's unique subunit organization, whereby each ring consists of eight different subunits in an arrangement that remains uncertain. Using single particle cryo‐EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7 Å resolution, which is the highest resolution asymmetric cryo‐EM structure to date. This revealed the existence of a two‐fold axis between its two rings. A subsequent two‐fold symmetrized map yielded a 4.0 Å resolution structure that evinces the densities of a large fraction of side‐chains, loops and insertions, which permitted unambiguous identification of all eight individual subunits. Independent biochemical near‐neighbor analysis supports our TRiC subunit arrangement. This allowed the optimization of a Cα backbone model of the entire TRiC complex from the homology models against the cryo‐EM density. A refined atomic model for one subunit showed ~95% of the dihedral angles in the allowable regions of the Ramachandran plot. Our model reveals an unevenly distributed positively charged wall lining the closed folding chamber of TRiC is strikingly different from those of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.Supported by Nanomedicine Development Center (PN1EY016525) and NCRR (P41RR02250).