biostudies-literatureCushing VICancer Research UKMedical Research Council2265https://www.ebi.ac.uk/biostudies/studies/S-EPMC10937634biostudies-literatureUnknown15(1)Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.Nature communicationsHigh-resolution cryo-EM of the human CDK-activating kinase for structure-based drug design.PMC10937634C37/A12011C37/A9335C37/A18784MR/V009354/1Cushing VIKoh AFKroll SHBJurgaityte KGreber BJBahl AKBarrett AGMFeng JBondke AAli SKotecha ABarbazanges MScheiper BfalseHigh-resolution cryo-EM of the human CDK-activating kinase for structure-based drug design.Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.2024-01-01T00:00:00Z2024 Mar2026-07-15T13:55:37.719Z2026-07-05T03:12:06.741ZS-EPMC109376343848068110.1038/s41467-024-46375-9