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Concept
Microcrystal electron diffraction
Résumé
Microcrystal electron diffraction, or MicroED, is a CryoEM method that was developed by the Gonen laboratory in late 2013 at the Janelia Research Campus of the Howard Hughes Medical Institute. MicroED is a form of electron crystallography where thin 3D crystals are used for structure determination by electron diffraction. Prior to this demonstration, macromolecular (protein) electron crystallography was only used on 2D crystals, for example.
The method was developed for structure determination of proteins from nanocrystals that are typically not suitable for X-ray diffraction because of their size. Crystals that are one billionth the size needed for X-ray crystallography can yield high quality data. The samples are frozen hydrated as for all other CryoEM modalities but instead of using the transmission electron microscope (TEM) in imaging mode one uses it in diffraction mode with an extremely low electron exposure (typically < 0.01 e−/Å2/s). The nano crystal is exposed to the diffracting beam and continuously rotated while diffraction is collected on a fast camera as a movie. MicroED data is then processed using traditional software for X-ray crystallography without the need for specialized software for structure analysis and refinement. Importantly, both the hardware and software used in a MicroED experiment are standard and broadly available.
The first successful demonstration of MicroED was reported in 2013 by the Gonen laboratory. The structure of lysozyme, a classic test protein in X-ray crystallography. This was the first time that a protein structure was determined from 3D crystals using electron diffraction. The Abrahams group independently reported collecting rotation electron diffraction data collection using a Medipix quantum area detector on lysozyme crystals but were unable to solve the structure.
Detailed protocols for setting up the electron microscope and for data collections have been published.
MicroED data is collected using transmission electron (cryogenic) microscopy.
Source officielle
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