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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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Optimizing Sample Preparation for Cryogenic Electron Microscopy

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Algorithmic robustness to preferred orientations in single particle analysis by CryoEM.

C O S Sorzano¹, D Semchonok², S-C Lin³

¹Biocomputing Unit, Centro Nacional de Biotecnologia (CNB-CSIC), Darwin, 3, Campus Universidad Autonoma, 28049 Cantoblanco, Madrid, Spain.

Journal of Structural Biology

|January 9, 2021

Summary

Preferred orientations in single particle analysis cryo-electron microscopy (cryoEM) can hinder high-resolution structure determination. Certain image processing algorithms are more robust to these issues, improving 3D reconstruction outcomes.

Keywords:

Electron microscopy Preferred orientations Single particle analysis

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Area of Science:

Structural biology
Biophysics
Cryo-electron microscopy

Background:

Preferred orientations in single particle analysis (SPA) are a significant challenge in cryo-electron microscopy (cryoEM).
This phenomenon often impedes the achievement of high-resolution structural models.
While grid preparation is a primary factor, algorithmic robustness is also critical.

Purpose of the Study:

To evaluate the impact of preferred orientations on cryoEM structure determination.
To demonstrate the differential robustness of image processing algorithms to preferred orientations.
To provide insights into overcoming common hurdles in SPA.

Main Methods:

Analysis of three distinct cryoEM datasets exhibiting preferred orientations.
Comparative assessment of different image processing algorithms for angular assignment and 3D reconstruction.
Evaluation of reconstruction quality and artifact presence.

Main Results:

Certain image processing algorithms show greater resilience to preferred orientations than others.
Demonstrated ability to achieve or improve 3D reconstructions in challenging datasets.
Highlighted specific algorithmic approaches that mitigate preferred orientation artifacts.

Conclusions:

Algorithm choice significantly impacts the success of 3D reconstruction in the presence of preferred orientations.
Robust algorithms can overcome limitations imposed by preferred orientations in SPA.
This work offers practical guidance for improving cryoEM structure resolution.