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Genetically encoded multimeric tags for subcellular protein localization in cryo-EM.

Herman K H Fung1, Yuki Hayashi2, Veijo T Salo1

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|November 6, 2023
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Summary
This summary is machine-generated.

This study introduces genetically encoded multimeric particles (GEMs) for precise intracellular protein localization using cryo-electron tomography (cryo-ET). This method enables time-controlled, label-free imaging of protein targets within their native cellular environment.

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Area of Science:

  • Cellular and Molecular Biology
  • Biophysics
  • Microscopy and Imaging

Background:

  • Cryo-electron tomography (cryo-ET) offers high-resolution, label-free imaging of cellular structures.
  • Localizing specific macromolecules within complex cryo-ET data remains a significant challenge.

Purpose of the Study:

  • To develop a novel, ligand-inducible labeling strategy for accurate intracellular protein localization using cryo-ET.
  • To enable automated detection and time-controlled visualization of target proteins in their native cellular context.

Main Methods:

  • Development of genetically encoded multimeric particles (GEMs) with recognizable structural signatures.
  • Ligand-induced coupling of GEMs to fluorescently tagged proteins of interest.
  • Application of convolutional neural networks for automated GEM detection in cryo-ET data.
  • Cryo-correlative fluorescence and cryo-ET imaging in human cells.

Main Results:

  • Demonstrated successful subcellular localization of both endogenous and overexpressed proteins in various organelles.
  • Validated the automated detection of GEMs in cryo-ET volumes.
  • Established methods for quantifying labeling specificity and efficiency.
  • Showcased time-controlled labeling to minimize impact on native protein function.

Conclusions:

  • GEMs provide a powerful tool for precise, label-free localization of intracellular proteins via cryo-ET.
  • The ligand-inducible system allows for temporal control, preserving native protein function.
  • This approach enhances the capabilities of cryo-ET for studying macromolecular assemblies in situ.