The crystal structure of the yeast Hsp90 complex with ATP and the co-chaperone p23/Sba1 reveals the architecture of the 'closed' state of Hsp90. The structure shows extensive inter-domain and inter-strand interactions, detailed conformational changes in the N-terminal domain upon ATP binding, and the structural basis for p23/Sba1 stabilization of the closed state. Contrary to expectations, the closed Hsp90 does not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the ATPase cycle. Hsp90 is essential for activating many regulatory and signaling client proteins, and its function depends on ATP binding and hydrolysis. The p23/Sba1 co-chaperone stabilizes the ATP-bound state, slowing ATP turnover and enhancing client protein activation. The structure confirms the ATPase-coupled molecular clamp mechanism and provides insights into ATP-dependent activation of Hsp90 client proteins. The Hsp90-p23/Sba1 complex contains a central Hsp90 dimer with symmetrically disposed p23/Sba1 molecules. The N-domain forms an intimate dimeric interaction, involving significant conformational changes. The lid segment swings through nearly 180 degrees, exposing a hydrophobic patch that stabilizes N-domain association. The middle segments move closer together, but do not make contact. The N-domain and middle segment interact, with the N-domain making extensive interactions with the middle segment. The nucleotide makes a single contact outside the N-domain, involving the γ-phosphate and the head group of Arg380. The structural basis for conformation-dependent recruitment of p23/Sba1 is shown, with multiple interfaces contributing to the interaction. The ATPase cycle is coupled to conformational changes, and mutations affecting these changes alter Hsp90 ATPase activity and client protein activation. The structure provides a firm basis for understanding the mechanism of client protein activation by Hsp90. The study was supported by the Wellcome Trust and Cancer Research UK.The crystal structure of the yeast Hsp90 complex with ATP and the co-chaperone p23/Sba1 reveals the architecture of the 'closed' state of Hsp90. The structure shows extensive inter-domain and inter-strand interactions, detailed conformational changes in the N-terminal domain upon ATP binding, and the structural basis for p23/Sba1 stabilization of the closed state. Contrary to expectations, the closed Hsp90 does not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the ATPase cycle. Hsp90 is essential for activating many regulatory and signaling client proteins, and its function depends on ATP binding and hydrolysis. The p23/Sba1 co-chaperone stabilizes the ATP-bound state, slowing ATP turnover and enhancing client protein activation. The structure confirms the ATPase-coupled molecular clamp mechanism and provides insights into ATP-dependent activation of Hsp90 client proteins. The Hsp90-p23/Sba1 complex contains a central Hsp90 dimer with symmetrically disposed p23/Sba1 molecules. The N-domain forms an intimate dimeric interaction, involving significant conformational changes. The lid segment swings through nearly 180 degrees, exposing a hydrophobic patch that stabilizes N-domain association. The middle segments move closer together, but do not make contact. The N-domain and middle segment interact, with the N-domain making extensive interactions with the middle segment. The nucleotide makes a single contact outside the N-domain, involving the γ-phosphate and the head group of Arg380. The structural basis for conformation-dependent recruitment of p23/Sba1 is shown, with multiple interfaces contributing to the interaction. The ATPase cycle is coupled to conformational changes, and mutations affecting these changes alter Hsp90 ATPase activity and client protein activation. The structure provides a firm basis for understanding the mechanism of client protein activation by Hsp90. The study was supported by the Wellcome Trust and Cancer Research UK.