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Related Concept Videos

Immunogold Electron Microscopy01:20

Immunogold Electron Microscopy

Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.
Cryo-electron Microscopy01:28

Cryo-electron Microscopy

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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Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging
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Published on: July 12, 2022

A ferritin-based label for cellular electron cryotomography.

Qing Wang1, Christopher P Mercogliano, Jan Löwe

  • 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.

Structure (London, England : 1993)
|February 9, 2011
PubMed
Summary

Researchers developed a new ferritin label for electron cryotomography, enabling precise molecule identification in cells. This advancement overcomes current limitations in visualizing cellular structures at high resolution.

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Electron Cryotomography of Bacterial Cells
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Electron Cryotomography of Bacterial Cells

Published on: May 6, 2010

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Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging
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Electron Cryotomography of Bacterial Cells
14:23

Electron Cryotomography of Bacterial Cells

Published on: May 6, 2010

Area of Science:

  • Structural Biology
  • Cell Biology
  • Biotechnology

Background:

  • Electron cryotomography (cryo-ET) offers high-resolution structural insights into near-native biological specimens.
  • A significant limitation of cryo-ET is the absence of specific molecular labels for identification, unlike fluorescence microscopy's Green Fluorescent Protein (GFP).
  • Developing such a label is crucial for advancing molecular localization studies using cryo-ET.

Purpose of the Study:

  • To engineer and validate a novel, clonable label for electron cryotomography.
  • To create a molecular tag analogous to GFP for enhanced visualization in cryo-ET.
  • To enable unambiguous identification of molecules within cellular cryo-electron tomograms.

Main Methods:

  • Utilized the Escherichia coli ferritin FtnA protein as a basis for the novel label.
  • Engineered ferritin fusions with a membrane-targeting sequence for directed localization.
  • Applied electron cryotomography to visualize overproduced ferritin within E. coli cells and assessed localization accuracy.

Main Results:

  • Demonstrated that overproduced ferritin is readily visible and detectable using cryo-electron tomography.
  • Successfully directed the ferritin label to the cell membrane by fusing it with a membrane-targeting sequence.
  • Achieved labeling patterns with the ferritin tag via cryo-ET that closely matched those obtained using fluorescence microscopy with GFP.

Conclusions:

  • The ferritin FtnA protein serves as an effective and clonable label for electron cryotomography.
  • This ferritin label demonstrates efficient and faithful localization within cells.
  • The developed ferritin label represents a valuable new tool for precise molecular identification in cellular cryo-electron tomograms.