title: A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray.
abstract: 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 microm in diameter from the 1-2 microl 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 >1s.
authors.affiliation: Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA 23507, USA. whited@evms.edu
authors.forename: H D
authors.initials: HD
authors.lastname: White
authors.forename: K
authors.initials: K
authors.lastname: Thirumurugan
authors.forename: M L
authors.initials: ML
authors.lastname: Walker
authors.forename: J
authors.initials: J
authors.lastname: Trinick
mesh_terms: D055592:Biophysical Phenomena; D001703:Biophysics; D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods*; D004980:Ethane / Q000737:chemistry; D005615:Freezing; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007700:Kinetics; D008854:Microscopy, Electron / Q000379:methods*; D013997:Time Factors
chemical_list: D004980:Ethane