Protocol to study CCT-mediated folding of Gβ₅ by single-particle cryo-EM

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.