Overcoming the Challenges of Structural Virology with Cryo-EM

Cryo-electron microscopy (cryo-EM) is advancing structural virology in instances where purifying virus proteins is a challenge, protein homogeneity is difficult to achieve, and image averaging may be the only way to obtain structural information at high resolution.

These structural insights are revolutionizing antiviral drug discovery and vaccine design for newly emerging pathogens.

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The Burden of Viral Diseases

Viral diseases cause symptoms in humans such as spots, runny noses, swelling, and fever. They can also be fatal.

New viruses periodically emerge unpredictably and cause great personal and social harm. In the wake of recent outbreaks, such as HIV, COVID-19, Zika, and Ebola, structural virology studies have taken center stage. There is an urgent need to characterize virus structure and host immune response to identify and develop therapeutics and vaccines.

Structural Virology Tools

A major challenge for virologists is to understand the molecular basis of viral life cycles and to use this knowledge to design new anti-viral strategies.

The current understanding of viral life cycles comes from molecular biology combined with structural virology. Scientists use several techniques to determine protein structures:

Nuclear magnetic resonance (NMR) provides unique information about protein dynamics and interactions, but this method restricts atomic structure determination to small complexes with molecular weights below 40-50 kDa.
X-ray crystallography achieves atomic details for smaller viruses, but it is a challenge to visualize a larger multi-shelled virus structure, complexes between viruses, and antibodies or receptors.
With cryo-EM, researchers rapidly vitrify a cell, virus, molecular complex, or other structure to preserve samples in their natural state. Scientists use cryo-EM to produce higher-resolution images compared to other structural techniques because of modern electron detectors and image-processing. The number of cryo-EM images uploaded to the Protein Data Bank has boomed in recent years and the technique won its developers the 2017 Nobel Prize in Chemistry.

Researchers can use cryo-EM to determine a virus structure quickly, even when the biochemistry involved in virus preparation is challenging. Although it may not be generally applicable to every emerging pathogen, cryo-EM is now a routine tool to aid in rapid vaccine development.

Insights from the Field: Q&A with Dr. Georgios Skiniotis

Starting as a new professor at the University of Michigan, Georgios Skiniotis’s first task was to build a cryo-EM lab. Since then, he applied cryo-EM to study a number of complicated macromolecular machines, such as enzymes that operate as factories to assemble complex chemicals with antibiotic and anticancer properties, and pharmacologically important cell signaling systems, such as his work on G protein-coupled receptors (GPCRs) that contributed to Nobel Prize-winning research.

Now a professor at Stanford University, Skiniotis employs cryo-EM to obtain an unprecedented view of cell signaling complexes.

What is your background in cryo-EM?

As a graduate student at the European Molecular Biology Lab in Heidelberg, I used cryo-EM to study kinesin motors, capturing snapshots that show how kinesin “walks” along a microtubule. Later, as a postdoctoral researcher in the laboratory of Tom Walz at Harvard Medical School, I became familiar with single-particle EM analysis and applied the technique to study transmembrane cytokine receptor assemblies. This training provided the foundation to start my own lab applying and advancing cryo-EM.

The main theme of my lab is the structural biology of cell surface receptors that mediate intracellular signaling and communication. We employ primarily cryo-EM 3D reconstructions complemented by biochemistry, biophysics, and simulation methods to obtain a dynamic view into the macromolecular complexes carrying out these processes. The major focus of my research is GPCRs and their complexes with transducers such as G proteins, assemblies that represent signal relay systems and are involved in all aspects of human physiology. My lab used single-particle negative-stain EM to obtain the first low-resolution structures of a GPCR/G protein complex in different conformations and followed up with the first cryo-EM reconstructions of these complexes.

How does cryo-EM overcome the challenges involved in x-ray crystallography?

It has always been challenging to go after the structures of membrane proteins with x-ray crystallography. One of the main reasons is that to obtain x-ray structures, you need to generate high-quality crystals. Very often with membrane proteins, you cannot have adequate packing interactions between layers of your protein to make well-ordered crystals. For example, most GPCRs do not have large intracellular or extracellular domains to establish such contacts, and they are also very dynamic proteins, assuming different conformations that do not favor crystallogenesis. As a result, most crystallographic efforts depended on extensive engineering and were often unsuccessful. Cryo-EM does not depend on generating crystals, and an ensemble of distinct conformations can be captured and identified.

How has cryo-EM advanced structural biology?

It essentially removed the crystallization bottleneck to get a structure. That opens up so many possibilities. Smaller proteins or rigid complexes are relatively easy to crystallize, but most biological processes involve larger, complex, and dynamic protein assemblies assuming various conformations. With cryo-EM we can record the projections of such entities as they occur in solution or embedded in a membrane patch, and apply computational analysis to obtain high resolution 3D reconstructions.

What is your advice to researchers who want to use cryo-EM?

First ask yourself the question, do you know what the structure represents? What is the biological relevance? Also, if you want to do successful cryo-EM, you need to have very good biochemical control of what you are looking at. You can get structures, but you need to interpret these structures successfully. Whenever someone from my lab purifies a protein or complex, I ask them to set up functional assays to learn if the purified protein is indeed functional and at what level before they put a lot of effort into getting a structure. Yes, the structure will always tell you something, but it can also be misleading if you do not really know what you have in your hands.

Georgios Skiniotis is a Professor of Structural Biology, and of Molecular and Cellular Physiology at Stanford University, and, by courtesy, of Photon Science at SLAC National Accelerator Laboratory. He is also the Director of the Stanford Cryo-EM Center.