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The Hsp90 chaperone complex is responsible for the activation and maturation of a vast array of signaling proteins. Recently, there has been a leap in our understanding of the Hsp90 structure and molecular mechanisms involved in such regulation. This has opened a doorway to the underlying mechanisms of disease processes and how we may intervene in such mechanisms to improve prognosis. The focus of this Special Issue is on the recent structural and biochemical advances of Hsp90 that provide insights into the mechanistic activation of Hsp90 on client proteins. It will also include discussions on Hsp90 and its co-chaperones, and their potential as therapeutic targets against human diseases.