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.

蛋白质是动态分子，可以响应刺激进行快速的构象重排。这些结构变化通常对蛋白质功能至关重要，因此阐明时间依赖性构象景观一直是结构生物学的长期目标。为了利用单粒子冷冻 EM 方法的力量来实现“时间分辨”结构测定，我们开发了一种光耦合冷冻柱塞，可将笼配体的闪光光解与快速样品玻璃化相结合。“闪光柱塞”由高功率紫外线 LED 与聚焦光学器件和电动线性致动器组成，使用户能够在刺激传递后的可编程时间窗口内（短至数十毫秒）将蛋白质目标固定在玻璃冰中. 闪光柱塞是一个简单的，