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Related Concept Videos

Cryo-electron Microscopy01:28

Cryo-electron Microscopy

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Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...
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Transmission electron microscopy (TEM) can be used to determine the 3D structure of biological samples with the help of techniques such as electron microscope tomography and single-particle reconstruction. While single-particle reconstruction can examine macromolecules and macromolecular complexes in vitro conditions only, tomography permits the study of cell components or small cells in vivo.
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Microcrystal Electron Diffraction of Small Molecules
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Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED.

Emma Danelius1,2,3, Tamir Gonen4,5,6

  • 1Department of Biological Chemistry, University of California Los Angeles, Los Angeles, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 5, 2021
PubMed
Summary

Microcrystal Electron Diffraction (MicroED) is a rapid cryo-electron microscopy technique for determining molecular structures. This protocol details MicroED data collection and processing for high-resolution structure determination in under an hour.

Keywords:
3D Electron CrystallographyCryo-EMElectron DiffractionGrid PreparationMicroEDMicrocrystalNanocrystalProtein StructureTransmission Electron Microscopy

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Area of Science:

  • Structural Biology
  • Biophysics
  • Materials Science

Background:

  • Microcrystal Electron Diffraction (MicroED) is an emerging cryo-electron microscopy (cryo-EM) technique.
  • Over 70 protein, peptide, and small organic molecule structures have been determined using MicroED.
  • The method involves analyzing nanocrystalline samples using electron diffraction.

Purpose of the Study:

  • To provide a detailed protocol for MicroED data acquisition and processing.
  • To enable high-resolution structure determination of micro- and nanocrystalline samples.
  • To streamline the MicroED workflow for rapid structure elucidation.

Main Methods:

  • Sample preparation involves depositing micro- or nanocrystals in solution onto electron microscopy grids.
  • Data collection is performed using a cryo-electron microscope under cryogenic conditions.
  • Continuous rotation diffraction data are processed using standard X-ray crystallography software.

Main Results:

  • The protocol covers nanocrystal identification, microscope setup, and data collection/processing.
  • High-resolution structures can be determined from well-behaved crystals exhibiting high-resolution diffraction.
  • The entire MicroED process can be completed in under an hour for suitable samples.

Conclusions:

  • MicroED offers a rapid and effective method for high-resolution structure determination.
  • The outlined protocol facilitates the application of MicroED in various scientific disciplines.
  • This technique expands the capabilities of cryo-EM for analyzing crystalline materials.