Laboratory of Molecular Nanodynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
Acta Crystallogr D Struct Biol. 2022 Jul 1;78(Pt 7):883-889. doi: 10.1107/S205979832200554X. Epub 2022 Jun 14.
A novel approach to time-resolved cryo-electron microscopy (cryo-EM) has recently been introduced that involves melting a cryo sample with a laser beam to allow protein dynamics to briefly occur in the liquid, before trapping the particles in their transient configurations by rapidly revitrifying the sample. With a time resolution of just a few microseconds, this approach is notably fast enough to study the domain motions that are typically associated with the activity of proteins but which have previously remained inaccessible. Here, crucial details are added to the characterization of the method. It is shown that single-particle reconstructions of apoferritin and Cowpea chlorotic mottle virus from revitrified samples are indistinguishable from those from conventional samples, demonstrating that melting and revitrification leaves the particles intact and that they do not undergo structural changes within the spatial resolution afforded by the instrument. How rapid revitrification affects the properties of the ice is also characterized, showing that revitrified samples exhibit comparable amounts of beam-induced motion. The results pave the way for microsecond time-resolved studies of the conformational dynamics of proteins and open up new avenues to study the vitrification process and to address beam-induced specimen movement.
一种新的时间分辨冷冻电子显微镜（cryo-EM）方法最近被引入，该方法涉及用激光束熔化冷冻样品，以使蛋白质动力学在液体中短暂发生，然后通过快速重结晶样品来捕获颗粒在其瞬态构型中。该方法的时间分辨率仅为几微秒，速度快到足以研究通常与蛋白质活性相关但以前无法访问的结构域运动。在这里，对该方法的特征进行了关键补充。结果表明，从重结晶样品中获得的脱铁蛋白和豇豆花叶病毒的单颗粒重建与传统样品没有区别，这表明融化和重结晶使颗粒保持完整，并且在仪器提供的空间分辨率内它们不会发生结构变化。还对快速重结晶如何影响冰的性质进行了表征，结果表明重结晶样品表现出相当量的束致运动。这些结果为蛋白质构象动力学的微秒时间分辨研究铺平了道路，并开辟了新的途径来研究玻璃化过程和解决束致样品运动问题。
