Electron cryomicroscopy as a powerful tool in biomedical research

Abstract

A human cell is a precisely regulated system that relies on the complex interaction of molecules. Structural insights into the cellular machinery at the atomic level allow us to understand the underlying regulatory mechanism and provide us with a roadmap for the development of novel drugs to fight diseases. Facilitated by recent technological breakthroughs, the Nobel prize-winning technique electron cryomicroscopy (cryo-EM) has become a versatile and extremely powerful tool to solve routinely near-atomic resolution three-dimensional protein structures. Consequently, it has become the focus of attention for structure-based drug design. In this review, we describe the basics of cryo-EM and highlight its growing role in biomedical research. Furthermore, we discuss latest developments as well as future perspectives.

Keywords: Biological macromolecule; Biomedical research; Cryo-EM; Drug design; Electron cryomicroscopy.

Fig. 1

The most common cryo-EM techniques. Schematic drawing in (a) to (d) illustrates principles of the most popular cryo-EM methods in the upper panel and shows corresponding raw data in the lower panel. (a) Single-particle cryo-EM: particles are embedded in a thin layer of amorphous ice. Resulting representative class averages are shown as insets on the right. Scale bar, 50 nm. (b) Single-particle negative-stain EM: particles are embedded in a layer of heavy metal salts to increase the weak contrast of biological materials. Resulting representative class averages are shown as insets on the right. Scale bar, 50 nm. (c) Micro-ED: small 3-D crystals are hit with a focused electron beam and diffraction patterns are recorded at different tilt angles. Inset shows a small section of the diffraction image with individual diffraction spots at higher magnification. The electron diffraction image was kindly provided by T. Gonen, Janelia Research Campus. (d) Cryo-ET: the specimen is tilted within the microscope and images at different angles are recorded. A tomographic slice shows the cellular periphery with microtubule bundles (black arrows) and plasma membrane (green arrows). (e) Resolution range coverage of various methods in structural biology. Color code used for the TEM-based methods corresponds to (a)–(d). Yellow: single-particle analysis; orange: electron crystallography/micro-ED; red: electron tomography

Fig. 2

High-resolution cryo-EM as tool for structure-based drug design. (A) Electron density map of the 1.8-Å structure of glutamate dehydrogenase, showing that single-particle cryo-EM is capable of achieving atomic resolution. Subunits of the homo-hexameric enzyme are colored in magenta, pink, cyan, and three different green hues. [EMD-8194]. (B) Visualization of the density for the cyclic peptide jasplakinolide (yellow) in the cryo-EM map of the non-canonical actin PfAct1 from the malaria-causing parasite Plasmodium falciparium, demonstrating the potential of cryo-EM in structure-based drug design. The actin filament is shown in light blue with central subunits colored in dark blue, magenta and cyan. [EMD-3805]. (C) The Volta phase plate has revolutionized the EM field by providing unprecedented contrast for biological specimen without the need of defocusing. The introduction of additional phase shift greatly enhances the phase contrast. Scale bar, 10 nm

Fig. 3

Cryo-EM structures of filamentous proteins and their biomedical relevance. (A) Tobacco mosaic virus is one of the most widespread viruses around the world, being a prime example for plant pathogens that can have far-reaching consequences for the economy as well as food supply. [EMD-2842]. (B) Microtubules are not only core components of the cytoskeleton, but are also essential in axonal transport along neurons, constituting the track for cargo-transporting motor proteins. [EMD-8322]. (C) Tau filaments are neurodegenerative deposits that are found in the brains of AD patients. [EMD-3741]. (D) The interaction of F-actin and myosin filaments is responsible for muscle contraction. Malfunctions can cause myopathies. [EMD-8165]

Fig. 4

Selected examples of biomedically relevant cryo-EM structures. (A) Cryo-EM structure of the trimeric envelope glycoprotein of HIV. It was solved in complex with two neutralizing antibody Fab fragments. [EMD-3308]. (B) Cryo-EM structure of the anthrax protective antigen pore from Bacillus anthracis. [EMD-6224]. (C) Cryo-EM structure of TcdA1 from Photorhabdus luminescens in its prepore state. [EMD-3645]. (D) Cryo-EM structure of the ryanodine receptor RyR1. Map at low threshold (transparent) is shown to visualize the nanodisc (blue arrow), which stabilizes the transmembrane helices of RyR1. [EMD-2751]. Individual subunits are depicted in various colors. The hetero-trimeric HIV envelope glycoprotein in A can be divided into the gp120 trimer (yellow, orange, and red) and gp41 trimer (pink, blue, and cyan). Bound Fab fragments (green) are indicated by black arrows. Heptameric anthrax protective antigen pore (yellow, orange, light red, dark red, magenta, cyan, and green) in (B), pentameric TcdA1 (yellow, orange, red, green and blue) in (C), and tetrameric RyR1 (yellow, orange, red, and green) in D