This study presents high-resolution cryo-EM structures of the human CDK-activating kinase (CAK) in its free and nucleotide-bound states, as well as in complex with 15 inhibitors, achieving up to 1.8 Å resolution. These structures provide detailed insights into inhibitor interactions and water molecule networks in the active site of cyclin-dependent kinase 7 (CDK7), offering a foundation for rational drug design. The CAK is a master regulator of cell growth and division, and its inhibition is a promising strategy for cancer and antiviral therapies. The study highlights the importance of structural data in understanding inhibitor selectivity and improving the design of next-generation therapeutics. The cryo-EM approach enables high-resolution structural analysis of dynamic systems, which is crucial for CDKs due to their conformational dependence on compound binding. The study demonstrates the feasibility of using cryo-EM for structure-based drug design, with a workflow that allows for high-throughput determination of inhibitor-bound CAK complexes. The results establish cryo-EM as a powerful tool for drug discovery, providing insights into the structural basis of CDK7 inhibitor selectivity and guiding the development of more effective therapeutics. The study also reveals the importance of water molecules in inhibitor binding and highlights the potential of cryo-EM in understanding the mechanisms of CDK7 selectivity. The findings contribute to the broader understanding of CDK biology and the development of targeted therapies for cancer and other diseases.This study presents high-resolution cryo-EM structures of the human CDK-activating kinase (CAK) in its free and nucleotide-bound states, as well as in complex with 15 inhibitors, achieving up to 1.8 Å resolution. These structures provide detailed insights into inhibitor interactions and water molecule networks in the active site of cyclin-dependent kinase 7 (CDK7), offering a foundation for rational drug design. The CAK is a master regulator of cell growth and division, and its inhibition is a promising strategy for cancer and antiviral therapies. The study highlights the importance of structural data in understanding inhibitor selectivity and improving the design of next-generation therapeutics. The cryo-EM approach enables high-resolution structural analysis of dynamic systems, which is crucial for CDKs due to their conformational dependence on compound binding. The study demonstrates the feasibility of using cryo-EM for structure-based drug design, with a workflow that allows for high-throughput determination of inhibitor-bound CAK complexes. The results establish cryo-EM as a powerful tool for drug discovery, providing insights into the structural basis of CDK7 inhibitor selectivity and guiding the development of more effective therapeutics. The study also reveals the importance of water molecules in inhibitor binding and highlights the potential of cryo-EM in understanding the mechanisms of CDK7 selectivity. The findings contribute to the broader understanding of CDK biology and the development of targeted therapies for cancer and other diseases.