The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the “power stroke” that ejects substrate into the folding chamber. Display omitted ► Single-particle cryo-EM identifies seven ATP-bound structures of GroEL chaperonin ► Analysis of structures suggests trajectory of domain movements triggered by ATP binding ► The structures delineate a mechanism for mechanical unfolding of polypeptide substrate ► Model suggests an intermediate conformation binding substrate and GroES simultaneously Cryo-EM analysis reveals a series of conformational steps through which GroEL binds polypeptide substrates, stretches and unfolds them, and then moves them into a folding chamber that promotes adoption of the native state.