In eukarya, chaperones Hsp70 and ​Hsp90 act coordinately in the folding and maturation of a range of key proteins with the help of several co-chaperones, especially ​Hop. Although biochemical data define the ​Hop-mediated Hsp70–​Hsp90 substrate transfer mechanism, the intrinsic flexibility of these proteins and the dynamic nature of their complexes have limited the structural studies of this mechanism. Here we generate several complexes in the Hsp70/​Hsp90 folding pathway (​Hsp90:​Hop, ​Hsp90:​Hop:Hsp70 and ​Hsp90:​Hop:Hsp70 with a fragment of the client protein ​glucocorticoid receptor (​GR-LBD)), and determine their 3D structure using electron microscopy techniques. Our results show that one ​Hop molecule binds to one side of the ​Hsp90 dimer in both extended and compact conformations, through ​Hop domain rearrangement that take place when Hsp70 or Hsp70:​GR-LBD bind to ​Hsp90:​Hop. The compact conformation of the ​Hsp90:​Hop:Hsp70:​GR-LBD complex shows that ​GR-LBD binds to the side of the ​Hsp90 dimer opposite the ​Hop attachment site.