Reaction-based cryo-EM resolves the continuous conformational spectrum of CTP synthase catalysis.

Capturing enzymes under native turnover conditions remains a grand challenge in structural biology. Here, we develop a reaction-based cryo-electron microscopy (cryo-EM) strategy that directly samples Drosophila melanogaster cytidine triphosphate synthase (CTPS) from actively catalyzing mixtures containing only natural substrates and allosteric effectors. By integrating reaction-based sampling with three-dimensional variability analysis, we resolve a continuous conformational spectrum of CTPS during catalysis. This approach visualizes the chemical progression of 4-phosphoryl-uridine triphosphate (4Pi-UTP) formation and reveals a previously unobserved co-occupancy pattern of adenosine triphosphate (ATP) and CTP at the reaction end point. Cross-validation with nonhydrolyzable ATP analogs demonstrates that UTP phosphorylation by ATP, rather than mere ATP binding, shifts the conformational ensemble toward more closed states. Our findings establish reaction-based cryo-EM as a framework for resolving chemically annotated conformational ensembles without predefining a single trapped intermediate.