Martel, J. M. J.; Caramello, N.; Coquille, S.; Mathieu, E.; Petit, L.; Jacquet, P.; Appolaire, A.; Leonarski, F.; Olieric, V.; Wang, M.; Madern, D.; Royant, A.; Engilberge, S.

Time-resolved crystallography offers a window into the transient, atomic-scale structural changes that underlie biological function. Here we introduce Time-Resolved Functional Rotation Crystallography (TR-FRX), a method that enables molecular-level studies of ligand binding and enzymatic catalysis using single protein crystals at room temperature. Unlike many state-of-the-art time-resolved methodologies, TR-FRX relies on standard rotation-based X-ray data collection and does not require serial sample delivery strategies. By dispensing nanoliter-scale droplets of ligand or substrate directly onto crystals mounted in conventional holders, TR-FRX captures real-time structural snapshots while maintaining experimental accessibility. As a proof-of-concept, we first monitor the binding of N-acetylglucosamine to hen egg-white lysozyme, demonstrating that TR-FRX can reveal ligand recognition in crystallo on sub-second timescales. We then resolve the bidirectional catalytic mechanism of a prototypical enzyme from the tricarboxylic-acid-cycle, revealing sequential cofactor and substrate binding, catalytic loop dynamics, and substrate stabilization across time points spanning from 137 milliseconds to minutes. Requiring only micrograms of protein and standard beamline infrastructure, TR-FRX provides an accessible methodology for capturing transient enzymatic states and enables 100-ms timescale studies at room temperature on single protein crystals.