4.0 Å Cryo-EM Structure of the Mammalian Chaperonin: TRiC/CCT

TRiC is a eukaryotic chaperonin essential for de novo folding of ∼10% newly synthesized cytosolic proteins, many of which cannot be folded by other cellular chaperones. Unlike prokaryotic and archael chaperonins, each of its two rings consists of eight unique, but similar subunits. Using single particle cryo-EM, we determined the mammalian TRiC structure without any symmetry imposition at 4.7 A resolution, which is the highest resolution asymmetric cryo-EM reconstruction to date. An analysis of this map allowed us to elucidate the relative orientation of the two rings and the two-fold symmetry axis location between them. A subsequent two-fold symmetrized map yielded a 4.0 A structure, in which a large fraction of side chains and structural elements including loops and insertions appear as visible densities. These features permitted unambiguous identification of all eight individual subunits, despite their similarity. A Cα backbone model of the entire TRiC complex was subsequently refined from initial homology models against the cryo-EM density based on our subunit identification. A refined all-atom model for a single subunit showed ∼95% of the dihedral angles in the allowable regions of the Ramachandran plot. Our model reveals that the cavity walls of TRiC exhibit an overall positively charged surface property, the opposite of GroEL. The interior surface chemical properties likely play an important role for TRiC's unique substrate specificity.