CAGE-TRX expands the scope of time-resolved crystallography through genetically encoded active-site photocaging

We present CAGE-TRX, a broadly applicable time-resolved strategy for release-quench-probe cryocrystallography and release-probe room temperature serial crystallography. These workflows enable light-triggered control of enzyme activity via genetically encoded photocaged amino acids. By decoupling reaction initiation from substrate design, this approach allows synchronized catalysis in crystallo and the capture of transient intermediates. Using β-lactamases as model systems, we demonstrate efficient decaging, restoration of activity, and structural visualization of reaction intermediates.