摘要Drug discovery is a crucial part of human healthcare and has dramatically benefited human lifespan and life quality in recent centuries,however,it is usually time-and effort-consuming.Structural biology has been demonstrated as a powerful tool to accelerate drug development.Among different techniques,cryo-electron microscopy(cryo-EM)is emerging as the mainstream of structure determination of biomacromolecules in the past decade and has received increasing attention from the pharmaceutical industry.Although cryo-EM still has limitations in resolution,speed and throughput,a growing number of innovative drugs are being developed with the help of cryo-EM.Here,we aim to provide an overview of how cryo-EM techniques are applied to facilitate drug discovery.The development and typical workflow of cryo-EM technique will be briefly introduced,followed by its specific applications in structure-based drug design,fragment-based drug discovery,proteolysis targeting chimeras,antibody drug development and drug repurposing.Besides cryo-EM,drug discovery innovation usually involves other state-of-the-art techniques such as artificial intelligence(AI),which is increasingly active in diverse areas.The combination of cryo-EM and AI provides an opportunity to minimize limitations of cryo-EM such as automation,throughput and interpretation of medium-resolution maps,and tends to be the new direction of future development of cryo-EM.The rapid development of cryo-EM will make it as an indispensable part of modern drug discovery.