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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: Jun 7, 2025

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Published on: April 11, 2025

RNA sample optimization for cryo-EM analysis.

Xingyu Chen¹, Liu Wang², Jiahao Xie¹

¹The State Key Laboratory of Biotherapy, Department of Geriatrics and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China.

Nature Protocols

|November 15, 2024

Summary

This summary is machine-generated.

This study presents a protocol for optimizing RNA sample preparation for cryogenic electron microscopy (cryo-EM). It details methods for improving RNA structure determination and understanding RNA dynamics.

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Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Published on: January 9, 2015

Area of Science:

Structural Biology
Biochemistry
Molecular Biology

Background:

RNA molecules are crucial for biological processes, with their function dictated by their 3D structures.
Determining RNA 3D structures is challenging due to conformational heterogeneity and dynamics.
Cryogenic electron microscopy (cryo-EM) is emerging as a powerful tool for resolving RNA structures and dynamics.

Purpose of the Study:

To present a protocol for rigorous screening and iterative optimization of RNA preparation conditions for cryo-EM.
To facilitate high-resolution cryo-EM structure determination of challenging RNA targets.
To provide a starting point for further optimization of difficult RNA samples.

Main Methods:

Systematic screening and optimization of RNA preparation variables for cryo-EM analysis.
Application of cryo-EM data processing pipelines to resolve RNA dynamics and conformational changes.
Utilizing RNA modeling algorithms to generate atomic coordinates from cryo-EM density maps.

Main Results:

A comprehensive protocol is described for cryo-EM structure determination of RNAs.
The protocol includes methods for sample preparation, data processing, and atomic modeling.
It addresses the under-explored correlations between preparation conditions and cryo-EM outcomes.

Conclusions:

This protocol aids in overcoming challenges in RNA structure determination using cryo-EM.
It enables the resolution of RNA dynamics and conformational changes at moderate to high resolutions.
The protocol is designed for users with basic skills in RNA biochemistry, cryo-EM, and RNA modeling.