Development of Cryo-Electron Microscopy for High-Resolution Structure Determination of Biomolecules in Solution: 2017 Nobel Prize in Chemistry

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.9 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our ~2.8 Å and ~3.2 Å resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.

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Von der „Blobology“ zur atomaren Auflösung der Kryo-Elektronenmikroskopie

BIOspektrum ◽

10.1007/s12268-020-1480-5 ◽

2020 ◽

Vol 26 (7) ◽

pp. 710-713

Author(s):

Holger Stark

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Structure Determination ◽

Protein Complexes ◽

Pharmaceutical Research ◽

New Drugs ◽

Cryo Electron Microscopy ◽

High Resolution Structure ◽

First Time

AbstractIt took almost a century to develop electron microscopy into a powerful method for high-resolution structure determination of proteins. Technical improvements in microscopy, detector technology, and image processing software contributed to the exponential growth of high-resolution structures of protein complexes determined by cryo-electron microscopy in recent years. We now succeeded in breaking another resolution barrier in cryo-electron microscopy and for the first time in achieving true atomic resolution, where single atoms in the protein can indeed be visualized individually. These improvements in cryo-EM indicate that the method will continue to gain importance, not only as a method for structure determination but also in the development of new drugs in pharmaceutical research.

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Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins

FEBS Letters ◽

10.1016/j.febslet.2010.04.015 ◽

2010 ◽

Vol 584 (12) ◽

pp. 2539-2547 ◽

Cited By ~ 38

Author(s):

Yo Sonoda ◽

Alex Cameron ◽

Simon Newstead ◽

Hiroshi Omote ◽

Yoshinori Moriyama ◽

...

Keyword(s):

High Resolution ◽

Membrane Proteins ◽

Structure Determination ◽

High Resolution Structure ◽

Resolution Structure

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Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

10.1101/2021.06.15.448552 ◽

2021 ◽

Author(s):

Nicole Dimos ◽

Carl P.O. Helmer ◽

Andrea M. Chanique ◽

Markus C. Wahl ◽

Robert Kourist ◽

...

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Structural Biology ◽

Structure Analysis ◽

Cryo Electron Microscopy ◽

High Resolution Structure ◽

Fast Development ◽

Pharmaceutical Industries ◽

Exquisite Specificity ◽

Resolution Structure

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 Å resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

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