A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray

• Published in: Journal of structural biology Vol. 144; no. 1; pp. 246 - 252
• Main Authors: White, H.D., Thirumurugan, K., Walker, M.L., Trinick, J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-10-2003
• Subjects: Biophysical Phenomena; Biophysics; Cryoelectron Microscopy - instrumentation; Cryoelectron Microscopy - methods; Ethane - chemistry; Freezing; Image Processing, Computer-Assisted - methods; Kinetics; Microscopy, Electron - methods; Time Factors
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2003.09.027

We describe here a second generation apparatus for studying transient reaction conformations in macromolecules and their complexes by electron cryo-microscopy. Reactions are trapped by rapid freezing in times ranging from a few milliseconds to tens of seconds after initiation. Blotting of the electron microscope grid and freezing it in liquid ethane uses computer controlled microstepping motors. For the fastest time resolution, a blotted grid containing a thin film of one reactant is sprayed with small droplets containing a second reactant just before freezing. The spray is produced electrically (electrospray), which gives a dense cloud of droplets <1
μm in diameter from the 1–2
μl of solution required per grid. A second method in which two solutions are first mixed by turbulent flow and then blotted prior to freezing is used for reactions with time courses >1
s.