Chapter title |
Time-Resolved Cryo-electron Microscopy Using a Microfluidic Chip
|
---|---|
Chapter number | 4 |
Book title |
Protein Complex Assembly
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7759-8_4 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7758-1, 978-1-4939-7759-8
|
Authors |
Sandip Kaledhonkar, Ziao Fu, Howard White, Joachim Frank, Kaledhonkar, Sandip, Fu, Ziao, White, Howard, Frank, Joachim |
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. |
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