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

Clyde A. Smith; Ailiena O. Maggiolo; Chasity P. Janosko; Maura E. Charette; Nilakshi Paul; Márta Tóth; Guillermo A. Calero; Sierra M Carr; Silvia Russi; Sergei B. Vakulenko; Alexander Deiters; Aina E. Cohen

doi:10.64898/2026.06.26.734858

BiochemistrybioRxivJun 29, 2026

Preprint

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

Clyde A. Smith iD, Ailiena O. Maggiolo iD, Chasity P. Janosko, Maura E. Charette iD, Nilakshi Paul, Márta Tóth iD, Guillermo A. Calero iD, Sierra M Carr, Silvia Russi iD, Sergei B. Vakulenko iD, Alexander Deiters iD, Aina E. Cohen iD

Abstract

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 {beta}-lactamases as model systems, we demonstrate efficient decaging, restoration of activity, and structural visualization of reaction intermediates.

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