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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

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Published on: January 9, 2015

Three dimensional electron microscopy and in silico tools for macromolecular structure determination.

Subhomoi Borkotoky¹, Chetan Kumar Meena¹, Mohammad Wahab Khan¹

¹Centre for Bioinformatics, School of Life Sciences, Pondicherry University, Puducherry-605014, India.

|April 20, 2016

Summary

Three dimensional transmission electron microscopy (3DTEM) offers advanced macromolecule structure determination without crystallization. This review covers recent 3DTEM advancements and complementary in silico tools for refined molecular complex structures.

Keywords:

CTF correction cryo-EM segmentation single particle analysis

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Area of Science:

Structural biology
Biophysics
Molecular imaging

Background:

Electron microscopy (EM) has emerged as a powerful tool in structural biology, complementing X-ray crystallography and NMR.
Three-dimensional transmission electron microscopy (3DTEM) is particularly adept at determining the structures of large molecular machines.
3DTEM allows for structure determination of macromolecules in their native state, eliminating the need for protein crystallization.

Purpose of the Study:

To review recent advancements in three-dimensional transmission electron microscopy (3DTEM).
To discuss the associated computational tools used in conjunction with 3DTEM.
To highlight the benefits of 3DTEM for determining the structures of macromolecular complexes.

Main Methods:

Utilizing three-dimensional transmission electron microscopy (3DTEM) for high-resolution structure determination.
Integrating 3DTEM data with in silico computational methods.
Analyzing macromolecular structures in their native, non-crystallized forms.

Main Results:

3DTEM provides 3D structures of macromolecules with no inherent size limitations.
The technique enables structure determination in a native-like state, bypassing crystallization requirements.
Combining 3DTEM with in silico tools leads to more refined structures of molecular complexes.

Conclusions:

3DTEM is a sophisticated and versatile technique for molecular structure determination.
Advancements in 3DTEM and computational tools are enhancing structural biology.
This approach facilitates a deeper understanding of complex molecular machinery.