分子机器,如聚合酶,核糖体,或者蛋白酶体,完成复杂的任务,需要他们的环境的热能。他们通过限制沿着可能的构象变化路径的随机运动来实现这一点。这些变化通常是通过与不同的辅因子接触来指导的,最好将其与布朗棘轮进行比较。许多分子机器在其整个功能周期中经历了三个主要步骤,包括初始化,重复处理,和终止。通过低温电子显微镜(cryo-EM)已经阐明了这些主要状态中的几种。然而,这些机器的各个步骤本身是独特的多步骤过程,他们在时间上的协调仍然难以捉摸。为了通过低温EM测量这些短暂的中间事件,总反应时间需要缩短以富集各自的平衡前状态。这种方法称为时间分辨低温EM(trEM)。在这次审查中,我们总结了trEM的方法发展及其在一系列生物学问题中的应用。
Molecular machines, such as polymerases, ribosomes, or proteasomes, fulfill complex tasks requiring the thermal energy of their environment. They achieve this by restricting random motion along a path of possible conformational changes. These changes are often directed through engagement with different cofactors, which can best be compared to a Brownian ratchet. Many molecular machines undergo three major steps throughout their functional cycles, including initialization, repetitive processing, and termination. Several of these major states have been elucidated by cryogenic electron microscopy (cryo-EM). However, the individual steps for these machines are unique and multistep processes themselves, and their coordination in time is still elusive. To measure these short-lived intermediate events by cryo-EM, the total reaction time needs to be shortened to enrich for the respective pre-equilibrium states. This approach is termed time-resolved cryo-EM (trEM). In this review, we sum up the methodological development of trEM and its application to a range of biological questions.