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Related Experiment Video

Updated: Sep 22, 2025

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

Published on: May 16, 2017

Deciphering a hexameric protein complex with Angstrom optical resolution.

Hisham Mazal^1,2, Franz-Ferdinand Wieser^1,2,3, Vahid Sandoghdar^1,2,3

¹Max Planck Institute for the Science of Light, Erlangen, Germany.

|May 26, 2022

Summary

This summary is machine-generated.

Polarization-encoded cryogenic optical localization in three dimensions (polarCOLD) enhances protein binding site resolution and measurement yield. This advanced technique reconstructs complex molecular structures with angstrom precision, applicable to cell and membrane biology.

Keywords:

assembly correlative imaging cryogenic super-resolution fluorescence molecular biophysics none protein structure structural biology

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Last Updated: Sep 22, 2025

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Published on: December 30, 2016

Area of Science:

Biophysics
Structural Biology
Molecular Imaging

Background:

Cryogenic optical localization in three dimensions (COLD) resolves protein binding sites using fluorophore blinking.
COLD's reliance on differing fluorophore brightness limits measurement yield and resolved sites.
A need exists to improve resolution and yield for studying large molecular complexes.

Purpose of the Study:

To enhance the number of resolved binding sites and measurement yield in cryogenic optical localization.
To develop a method capable of resolving heterogenous subsets within large molecular complexes.
To achieve angstrom-resolution structural determination of complex biomolecules.

Main Methods:

Partial labeling combined with polarization encoding to identify single fluorophores during blinking.
Utilizing a particle classification scheme to resolve heterogenous subsets.
Reconstructing three-dimensional arrangements of molecular complexes using the developed polarCOLD method.

Main Results:

Demonstrated polarCOLD by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA).
Resolved six distinct sites in the hexamer protein Caseinolytic Peptidase B (ClpB) of *Thermus thermophilus* with angstrom resolution.
Showcased the method's capability in determining quaternary structures of large molecular complexes.

Conclusions:

PolarCOLD significantly extends the capabilities of COLD for high-resolution structural studies.
The method is compatible with fluorescent protein labeling, broadening its application in cell and membrane biology.
Combining polarCOLD with cryo-electron microscopy (cryoEM) offers new insights into biomolecular structural heterogeneities.