The complex eukaryotic chaperonin TRiC/CCT helps maintain cellular protein homeostasis, however, its assembly mechanism remains largely unknown. To address the subunit specificity in TRiC assembly, we express each of the individual yeast TRiC subunit in E. coli. Our cryo-EM structural study and biochemical analyses demonstrate that CCT1/2/6 can form TRiC-like homo-oligomeric double ring (HR) complex, however ATP-hydrolysis cannot trigger their ring closure; after deletion of the long N-terminal extension, CCT5 can form the closed double-ring structure; while CCT3/4/7/8 cannot form the HRs. It appears that CCT1 forms a HR in a unique spiral configuration, and ATP-hydrolysis can drive it to re-assemble with an inserted extra subunit-pair. Our data suggest that CCT5 could be the leading subunit in ATP-hydrolysis-driven TRiC ring closure. Moreover, we demonstrate that ADP is sufficient to trigger the assembly of the HRs and TRiC from the assembly intermediate micro-complex form. Our study reveals that through evolution, the more ancestral subunits may have evolved to take more responsibilities in TRiC ring assembly, and we propose a possible assembly mechanism of TRiC involving subunit-pair insertion. Collectively, our study gives hints on the structural basis of subunit specificity in TRiC assembly and cooperativity, beneficial for future TRiC-related therapeutic strategy development.
Nanomechanical properties of a Ni nanodot-patterned surface (NDPS) on a Si substrate were investigated using nanoindentation. The Ni NDPS was fabricated by thermal evaporation of Ni through a porous anodized aluminum oxide template onto a Si substrate. Plan-view transmission electron microscopy and nanobeam diffraction were used to characterize the Ni nanodot crystal structure. Scanning electron microscopy and atomic force microscopy were used to characterize the morphology and deformation of the Ni nanodots before and after nanoindentation. The elastic modulus and hardness of the Ni nanodots were found to be 159 ± 22 and 7.7 ± 1.0 GPa, respectively. The critical shear stress for initiating plastic deformation in the Ni nanodot was estimated to be 8.3 ± 1.0 GPa, which is close to the theoretical shear strength of 7.6 GPa in dislocation-free single crystal Ni.
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