Ligand Binding: recording events in the active and alternative binding sites using time-resolved crystallography

On a molecular level, biological function is closely linked to the dynamic processes of enzymes. However, capturing these conformational changes, especially during catalysis, has proven difficult using conventional experimental methods. Uniquely, time-resolved X-ray crystallography (TRX) can provide insight into these dynamic changes, but is often compounded with substantial experimental challenges. To surmount these challenges and enable TRX on typical beamlines, we have pioneered cryo-trapping TRX. Our SPITROBOT crystal plunger enables to move beyond canonical static imaging of proteins and instead capture short-lived reaction intermediates, that report on dynamic biochemical processes as they unfold. Here we aim to demonstrate that the next generation plunger ‘SPITROBOT-2’ enables reaction quenching within 25 ms and allows following the dynamic events during ligand binding and catalysis for several model systems.