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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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To be visualized by an electron microscope, either transmission or scanning, biological samples need to be fixed (stabilized) so the electron beam does not destroy them and dried thoroughly (desiccated/dehydrated) so the vacuum does not affect them. Fixation needs to be done as quickly as possible because the sample properties will start changing as soon as it is removed from its natural environment. For example, in a tissue sample, the oxygen levels begin decreasing, causing an altered...
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A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline
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Comprehensive microcrystal electron diffraction sample preparation for cryo-EM.

William J Nicolas1, Cody Gillman2,3, Sara J Weaver2

  • 1Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, CA, USA.

Nature Protocols
|December 20, 2024
PubMed
Summary
This summary is machine-generated.

This guide details Microcrystal Electron Diffraction (MicroED) sample preparation for diverse molecules and proteins. It offers optimization strategies for researchers using this advanced cryogenic electron microscopy (cryo-EM) technique.

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Area of Science:

  • Structural biology
  • Biophysics
  • Materials science

Background:

  • Microcrystal Electron Diffraction (MicroED) is a powerful cryogenic electron microscopy (cryo-EM) technique for determining crystal structures.
  • It is applicable to a wide range of samples, including small molecules, soluble proteins, and membrane proteins.
  • Expertise in MicroED sample preparation, data acquisition, and processing is not always readily available.

Purpose of the Study:

  • To provide a comprehensive guide on MicroED sample preparation.
  • To address commonly used methods for various sample categories.
  • To offer instructions and optimization strategies for MicroED sample preparation.

Main Methods:

  • Detailed protocols for MicroED sample preparation are presented.
  • Methods cover room temperature solid-state small molecules, soluble proteins, and membrane protein crystals.
  • Optimization strategies are included for unique crystal growth and preparation conditions.

Main Results:

  • The guide offers practical instructions for new users of MicroED.
  • It provides a framework for optimizing sample preparation for diverse crystalline materials.
  • The protocol is suitable for researchers with existing expertise in biochemistry, crystallography, and cryo-EM.

Conclusions:

  • This resource aims to improve accessibility to MicroED technology.
  • Effective sample preparation is crucial for successful MicroED structure determination.
  • The guide empowers researchers to overcome challenges in MicroED sample preparation.