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Cryo-electron Microscopy

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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Updated: May 23, 2025

Author Spotlight: Enhancing CryoEM Sample Preparation Using Graphene Monolayer on Microscopy Grids

Author Spotlight: Enhancing CryoEM Sample Preparation Using Graphene Monolayer on Microscopy Grids

Published on: November 10, 2023

The graphene-based affinity cryo-EM grid for the endogenous protein structure determination.

Sojin An^1,2, Eungjin Ahn^1,3, Tyler Koo¹

¹Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

Biorxiv : the Preprint Server for Biology

|March 10, 2025

Summary

Researchers developed the Graffendor (GFD) grid, a novel graphene-based tool for cryo-electron microscopy (cryo-EM). This innovation improves the capture and high-resolution structure determination of low-abundance endogenous protein complexes.

Keywords:

ALFA nanobody ALFA tag affinity grid electron cryo-microscopy electron cryo-tomography endogenous protein graphene grid

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Last Updated: May 23, 2025

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Published on: November 10, 2023

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Area of Science:

Structural Biology
Biophysics
Cryo-Electron Microscopy

Background:

Sample preparation remains a key challenge for high-resolution cryo-electron microscopy (cryo-EM).
Targeting low-abundance endogenous protein complexes requires advanced techniques for efficient capture and structural analysis.

Purpose of the Study:

To develop and validate a novel graphene-based affinity cryo-EM grid, the Graffendor (GFD) grid, for improved structural determination of challenging protein targets.
To demonstrate the GFD grid's capability in capturing both tagged and endogenous proteins for high-resolution cryo-EM analysis.

Main Methods:

Development of a one-step crosslinking batch-production method for GFD grids using ALFA nanobodies (GFD-A grid).
Application of GFD-A grids for capturing tagged β-galactosidase and endogenous yeast proteins (Pop6, RNase MRP, RNase P).
Cryo-EM data collection and structure determination of target proteins at high resolution.

Main Results:

The GFD-A grid successfully captured tagged proteins, enabling cryo-EM structure determination at 2.71 Å.
High-resolution cryo-EM structures of endogenous RNase MRP (3.3 Å from lysate, 3.6 Å from eluate) and RNase P (3.0 Å from lysate, 3.9 Å from eluate) were obtained.
Additional densities observed in lysate-derived structures suggest the capture of transient interactions, absent in eluate-derived structures.

Conclusions:

The Graffendor (GFD) grid provides a robust platform for investigating endogenous proteins using cryo-EM.
This method enhances the efficiency and resolution of cryo-EM studies for challenging targets, including those with transient interactions.
The GFD-A grid facilitates the structural analysis of low-abundance protein complexes, advancing the field of structural biology.