Time-Resolved Cryo-electron Microscopy Using a Microfluidic Chip
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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.
Key wordsTime-resolved Single-particle cryo-EM Microfluidic chip Spraying Short-lived intermediates heterogeneity
This research has been supported by the HHMI and NIH R01 GM29169 and GM55440 (to J.F.) and NIH AR40964 and NIH Fogarty Senior International Fellowship (to H.W.).
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