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Summary: The chaperonin CCT mediates folding of many cytosolic proteins, including G protein β subunits (Gβs). Here, we present a protocol for isolating Gβ5 bound to CCT and its co-chaperone PhLP1 and determining the CCT-mediated folding trajectory of Gβ5 using single-particle cryoelectron microscopy (cryo-EM) techniques. We describe steps for purifying CCT-Gβ5-PhLP1 from human cells, stabilizing the closed CCT conformation, preparing and imaging cryo-EM specimens, and processing data to recover multiple Gβ5 folding intermediates. This protocol permits visualization of protein folding by CCT.For complete details on the use and execution of this protocol, please refer to Sass et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.