Light-coupled cryo-plunger for time-resolved cryo-EM.

Abstract Proteins are dynamic molecules that can undergo rapid conformational rearrangements in response to stimuli. These structural changes are often critical to protein function, and thus elucidating time-dependent conformational landscapes has been a long-standing goal of structural biology. To harness the power of single particle cryo-EM methods to enable ‘time-resolved’ structure determination, we have developed a light-coupled cryo-plunger that pairs flash-photolysis of caged ligands with rapid sample vitrification. The ‘flash-plunger’ consists of a high-power ultraviolet LED coupled with focusing optics and a motorized linear actuator, enabling the user to immobilize protein targets in vitreous ice within a programmable time window – as short as tens of milliseconds – after stimulus delivery. The flash-plunger is a simple, inexpensive and flexible tool to explore short-lived conformational states previously unobtainable by conventional sample preparation methods.