Time-Resolved Cryo-electron Microscopy Using a Microfluidic Chip

Methods Mol Biol. 2018;1764:59-71. doi: 10.1007/978-1-4939-7759-8_4.

Abstract

With the advent of direct electron detectors, cryo-EM has become a popular choice for molecular structure determination. Among its advantages over X-ray crystallography are (1) no need for crystals, (2) much smaller sample volumes, and (3) the ability to determine multiple structures or conformations coexisting in one sample. In principle, kinetic experiments can be done using standard cryo-EM, but mixing and freezing grids require several seconds. However, many biological processes are much faster than that time scale, and the ensuing short-lived states of the molecules cannot be captured. Here, we lay out a detailed protocol for how to capture such intermediate states on the millisecond time scale with time-resolved cryo-EM.

Keywords: Microfluidic chip; Short-lived intermediates heterogeneity; Single-particle cryo-EM; Spraying; Time-resolved.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy / instrumentation*
  • Cryoelectron Microscopy / methods*
  • Kinetics
  • Microchip Analytical Procedures / methods*
  • Microfluidics / methods*
  • Molecular Conformation
  • Ribosomes / ultrastructure*