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Related Concept Videos

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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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Related Experiment Video

Updated: Jun 13, 2026

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
09:37

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Published on: August 15, 2014

Macromolecular crystal data phased by negative-stained electron-microscopy reconstructions.

Stefano Trapani1, Guy Schoehn, Jorge Navaza

  • 1Université de Montpellier 1, Montpellier, France. stefano.trapani@cbs.cnrs.fr

Acta Crystallographica. Section D, Biological Crystallography
|May 7, 2010
PubMed
Summary

Negative staining with electron microscopy and X-ray diffraction enables structural analysis of medium-sized proteins. This method successfully determined the crystal structures of type II dehydroquinase enzymes from Candida albicans and Streptomyces coelicolor.

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Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition
08:01

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Published on: December 22, 2011

Area of Science:

  • Structural biology
  • Biophysics
  • Biochemistry

Background:

  • Combining transmission electron microscopy (TEM) and X-ray diffraction (XRD) is typically restricted to large particles.
  • Cryo-electron microscopy (cryo-EM) has limitations in sample preparation and applicability.
  • Developing accessible methods for medium-sized protein structure determination is crucial.

Purpose of the Study:

  • To extend the applicability of TEM-XRD integration to medium-sized proteins.
  • To establish negative staining as a viable alternative to cryo-EM for structural studies.
  • To solve the crystal structures of specific dehydroquinase enzymes.

Main Methods:

  • Utilized negative staining, a widely applicable electron microscopy technique.
  • Integrated negative staining data with X-ray diffraction (XRD) data.
  • Employed molecular replacement for structure solution.

Main Results:

  • Successfully produced models of medium-sized proteins suitable for molecular replacement.
  • Determined the crystal structure of the dodecameric type II dehydroquinase from Candida albicans (approx. 190 kDa).
  • Solved the crystal structure of the orthologous Streptomyces coelicolor type II dehydroquinase.

Conclusions:

  • Negative staining coupled with XRD is effective for determining the structures of medium-sized protein complexes.
  • This approach expands the utility of electron microscopy and X-ray diffraction integration.
  • The solved structures provide insights into the function and evolution of type II dehydroquinase enzymes.