This study reports the high-resolution cryo-EM structures of the human CDK-activating kinase (CAK), a master regulator of cell growth and division, in its free, nucleotide-bound, and inhibitor-bound states. The structures were determined at up to 1.8 Å resolution, providing detailed insights into inhibitor interactions and water networks in the active site of cyclin-dependent kinase 7 (CDK7). The research focuses on 15 inhibitors, including pyrazolopyrimidine, pyrazolotriazine, and phenylaminopyrimidine-class compounds, to understand inhibitor selectivity and mechanisms contributing to it. The study establishes a methodological framework for using high-resolution cryo-EM in structure-based drug design, particularly for next-generation therapeutics targeting CAK. The results highlight the importance of conformational changes and water-mediated interactions in inhibitor binding and selectivity, offering a basis for rational drug design.This study reports the high-resolution cryo-EM structures of the human CDK-activating kinase (CAK), a master regulator of cell growth and division, in its free, nucleotide-bound, and inhibitor-bound states. The structures were determined at up to 1.8 Å resolution, providing detailed insights into inhibitor interactions and water networks in the active site of cyclin-dependent kinase 7 (CDK7). The research focuses on 15 inhibitors, including pyrazolopyrimidine, pyrazolotriazine, and phenylaminopyrimidine-class compounds, to understand inhibitor selectivity and mechanisms contributing to it. The study establishes a methodological framework for using high-resolution cryo-EM in structure-based drug design, particularly for next-generation therapeutics targeting CAK. The results highlight the importance of conformational changes and water-mediated interactions in inhibitor binding and selectivity, offering a basis for rational drug design.