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The GroEL/ES chaperonin system is required for the assisted folding of many proteins.How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood.Using symmetryfree, single-particle electron cryo-microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation.