The eukaryotic group II chaperonin, the chaperonin-containing t-complex polypeptide 1 (CCT), plays an important role in cytosolic proteostasis. It has been estimated that as much as 10% of cytosolic proteins interact with CCT during their folding process. CCT is composed of 8 different paralogous subunits. Due to its complicated structure, molecular and biochemical investigations of CCT have been difficult. In this study, we constructed an expression system for CCT from a thermophilic fungus, Chaetomium thermophilum (CtCCT), by using E. coli as a host. As expected, we obtained recombinant CtCCT with a relatively high yield, and it exhibited fairly high thermal stability. We showed the advantages of the overproduction system by characterizing CtCCT variants containing ATPase-deficient subunits. For diffracted X-ray tracking experiment, we removed all surface exposed cysteine residues, and added cysteine residues at the tip of helical protrusions of selected two subunits. Gold nanocrystals were attached onto CtCCTs via gold-thiol bonds and applied for the analysis by diffracted X-ray tracking. Irrespective of the locations of cysteines, it was shown that ATP binding induces tilting motion followed by rotational motion in the CtCCT molecule, like the archaeal group II chaperonins. When gold nanocrystals were attached onto two subunits in the high ATPase activity hemisphere, the CtCCT complex exhibited a fairly rapid response to the motion. In contrast, the response of CtCCT, which had gold nanocrystals attached to the low-activity hemisphere, was slow. These results clearly support the possibility that ATP-dependent conformational change starts with the high-affinity hemisphere and progresses to the low-affinity hemisphere.