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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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A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

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Tools for visualizing and analyzing Fourier space sampling in Cryo-EM.

Philip R Baldwin¹, Dmitry Lyumkis²

¹The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA, 92037, USA.

Progress in Biophysics and Molecular Biology

|July 10, 2020

Summary

Understanding cryo-electron microscopy (cryo-EM) orientation distributions is crucial. We introduce a tool to assess how projection views affect reconstruction quality and resolution measures, enhancing data interpretation.

Keywords:

FSC Reconstruction Resolution SCF Sampling

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Area of Science:

Structural Biology
Biophysics
Computational Biology

Background:

A comprehensive understanding of orientation distribution's impact on cryo-EM reconstructions is lacking.
Assessing view sets is necessary to understand their effect on experimental reconstructions.

Purpose of the Study:

To investigate how orientation distribution systematically alters resolution measures in cryo-EM.
To introduce and validate the sampling compensation factor (SCF) for evaluating cryo-EM data collection geometry.
To develop a user-friendly tool for cryo-EM practitioners to analyze orientation distributions.

Main Methods:

Introduced the sampling compensation factor (SCF) to quantify the effect of collection geometry on spectral signal-to-noise ratio (SSNR).
Demonstrated that Fourier space sampling on large spherical surfaces is equivalent to knowing projection views.
Developed a graphical user interface (GUI) to visualize and analyze experimental orientation distributions.

Main Results:

The SCF geometrical factor can be calculated from spherical surfaces in Fourier space.
The GUI provides plots of projection directions, spherical sampling, SCF values, empty Fourier space fraction, and sampling histograms.
Incorporating a fixed tilt angle can improve view distribution and Fourier space sampling.

Conclusions:

A simplified conception of sampling and the developed GUI tool aid in understanding projection direction effects on cryo-EM reconstructions.
The SCF and associated tools offer a valuable complement to existing methods for analyzing cryo-EM data quality.
This work advocates for critical assessment of orientation distributions to improve cryo-EM reconstruction outcomes.