Cryo-Electron Microscopy: Guiding the Next Generation of Therapeutics With Complex Insights

Showcasing the incredible collaborative spirit of the scientific community, researchers across the world came together in 2020 to find solutions to the coronavirus pandemic. Now, this research continues as we strive to combat coronavirus variants and prepare for the next pandemic. Among these scientists is Assistant Professor Stephen Brohawn of UC Berkeley, who used cryo-electron microscopy (cryo-EM) to investigate the SARS-CoV-2 3a protein. Through the intensive work of his lab, as well as collaborations with scientists around the world, Brohawn and his team were able to determine the structure of 3a ion channel to 2.1 Å resolution—a remarkable feat given the difficulty and small size of this membrane protein.

Physiology and structural biology

Medicine has been driven by the observation of physiological systems at increasingly smaller scales. Microscopes allowed us to directly observe cells and pathogens, and, at the same time, monitor how various treatments would impact these in vitro and in vivo. Once we reached the limits of the light microscope, we began to utilize electron microscopy to view cells at even smaller scales, clearly visualizing not just their surfaces but also their interior organelles and structures. Through these observations we began to better understand cell-pathogen interactions, and how we might disrupt them to protect ourselves. Crystallography propelled us several steps ahead to the structural analysis of individual proteins. These fundamental insights allowed us to understand how proteins’ structure correlates to their function within the body, and how disruption of these structures can lead to physiological dysfunction.

A highly established technique, crystallography can be used to determine the structure of complicated proteins, and even some small protein complexes, but there are inherent limits to the technique. As is evident from the name, crystallography relies on fairly large crystals of the sample protein in order to produce a diffraction pattern. Crystallization becomes increasingly more challenging with larger and more hydrophobic samples. This means that systems like membrane proteins are particularly challenging for crystallography, as they have large hydrophobic regions that are designed to be embedded in a lipid membrane. Various artificial structures were developed to mimic the membrane environment and stabilize the protein, such as detergent micelles and lipidic cubic phase media, but these only serve to make crystallization even more challenging.

Fundamentally, this leaves a gap in our observational capability. We can view cells, and we can view many of their individual proteins, but we cannot yet bridge these scales. We can’t see how proteins act in complexes, or in vivo. If we could practically observe the full physiological scale from cell to atomic structure, we could generate highly effective and targeted treatments for diseases and disorders. Now, advances in cryo-EM are promising to bridge this divide between cellular and molecular biology.

Cryo-electron microscopy in structural biology

Cryo-EM describes a group of techniques, including single particle analysis and cryo-tomography, that are performed at cryogenic temperatures on specialized transmission electron microscopes (cryo-TEMs). Single particle analysis is by far the most popular cryo-EM technique, so much so that the term “cryo-EM” is often used synonymously with single particle analysis.

In cryo-EM, aqueous samples are first rapidly frozen, or vitrified, fixing the proteins in a thin layer of amorphous ice. Without the disruptive formation of crystalline ice, the suspended proteins are essentially locked in their native-state confirmations. The samples are then transferred to the cryo-TEM, where hundreds of individual protein molecules are imaged. Since the samples are oriented randomly in solution, the images provide 2D snapshots, or projections, of the protein from various angles. These images are then computationally recombined into a 3D representation of the sample; notably the higher the symmetry of the sample, the easier it is to create this reconstruction. With the advent of modern electron detectors, stable energy filters, and better electron sources, the resolution of cryo-EM structures can reliably reach < 3 Å, and atomic-resolution structures are even being reported for standard protein samples.

Since cryo-EM images individual proteins, there is no need for crystallization, which makes the technique ideally suited for samples that have historically been challenging for crystallography. This includes membrane protein and large complexes, both of which have difficulty forming enough interfaces to conform to a crystal lattice. Cryo-EM is also highly amenable to the methods used for membrane protein stabilization, such as lipid nanodiscs and detergent micelles.

SARS-CoV-2 3a protein: cryo-EM in practice

As the world continues to grapple with COVID-19 for a second year in a row, it is becoming increasingly clear that coronavirus-related illnesses are an unavoidable fixture of our future. The discovery and analysis of the SARS-CoV-2 spike protein showcased the immense power of structural biology and the critical role it plays in the development of highly effective vaccines. Now, researchers are racing to expand our understanding of coronaviruses in order to keep pace with the emergence of new disease variants. This includes not just the rapid structural analysis of variant spike proteins, but also the investigation of other coronavirus proteins that might serve as additional therapeutic and/or vaccine targets.

Cryo-EM map of the 3a dimer in MSP1E3D1 nanodiscs at 2.1-Å nominal resolution. Credit: David Kern, UC Berkeley.

Among these potential targets is the large 3a protein encoded by the open reading frame 3a (ORF3a) region of the coronavirus genome. 3a is a putative ion channel that is found in infected cells and is potentially associated with cell death as well as acute respiratory symptoms in individuals with the disease. As a membrane protein, 3a is inherently difficult to isolate and crystallize due to its hydrophobicity, making it an ideal target for investigation with cryo-EM. Researchers from the Brohawn and Bautista groups at UC Berkeley rose to the challenge, publishing the cryo-EM structure of 3a in a recent Nature Structural & Molecular Biology article. By utilizing lipid nanodiscs, they were able to isolate individual 3a proteins for single particle analysis, determining its structure to 2.1 Å resolution. This level of detail enabled them to visualize both an inner polar cavity as well as external hydrophilic grooves, two potential ion transduction regions of therapeutic interest. The prevalence of 3a-like protein across coronaviruses makes these observations an exciting first step toward alternative, targeted treatments for SARS-CoV-2 and similar diseases.

The future of structural biology

Cryo-electron microscopy is revolutionizing structural biology, supporting established methods with new insights that were long considered to be untenable. As researchers begin to bridge the gap between cellular and molecular insights, they can conceptualize how protein function relates to cellular behavior and ultimately physiology. These observations can lead to the development of highly precise, targeted treatments for diseases and disorders, as we are already beginning to see in the battle against coronaviruses. It is an exciting, pivotal time in structural biology, and cryo-EM is shaping up to be a major player.

About the authors: 

Abhay Kotecha is Manager, Application Scientists at Thermo Fisher Scientific.
Alex Ilitchev is Lead Scientific Editor at Thermo Fisher Scientific.