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Time-Resolved Cryo-electron Microscopy Using a Microfluidic Chip

  • Sandip Kaledhonkar
  • Ziao Fu
  • Howard White
  • Joachim Frank
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1764)

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.

Key words

Time-resolved Single-particle cryo-EM Microfluidic chip Spraying Short-lived intermediates heterogeneity 

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Sandip Kaledhonkar
    • 1
  • Ziao Fu
    • 2
  • Howard White
    • 3
  • Joachim Frank
    • 1
    • 4
  1. 1.Department of Biochemistry and Molecular BiophysicsColumbia UniversityNew YorkUSA
  2. 2.Integrated Program in Cellular, Molecular and Biomolecular StudiesColumbia University College of Physicians and SurgeonsNew YorkUSA
  3. 3.Physiological SciencesEastern Virginia Medical SchoolNorfolkUSA
  4. 4.Department of Biological SciencesColumbia UniversityNew YorkUSA

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