What is cryo-electron microscopy?

Cryo-electron microscopy, or cryo-EM, is a mainstay in structural biology. It is a sophisticated imaging technique designed to allow the visualization of biological molecules close to their native state. Crucially, this requires no dyes or fixatives used in room temperature microscopy, which may alter the specimen’s properties. Cryo-EM’s revolutionary capacity for helping determine the structures of large biomolecules and complexes at molecular resolution make it an essential tool in biology and medicine. Previously, achieving these results was challenging or impossible using conventional methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.

The Basics of Cryo-EM

Cryo-EM involves rapidly cooling a sample to cryogenic temperatures (usually using liquid ethane at approximately -188°C) to vitrify the water around the sample. Vitrification is a process where water transitions into a glass-like solid without forming ice crystals, which could disrupt the molecular structure of the sample. This rapid cooling is essential to preserve the native structure and to prevent artifacts that might arise from crystalline ice formation.

The frozen samples trapped in vitreous water are then imaged in a transmission electron microscope. Electrons are accelerated and focused into a beam that passes through the sample. Because electrons have much shorter wavelengths than visible light, they can reveal structures at much finer resolutions. The interaction of the electrons with the sample produces an image that is magnified and projected onto a detector, typically a charge-coupled device (CCD) or a direct electron detector.

Advantages of Cryo-EM

One of the key advantages of cryo-EM is its ability to study heterogeneous samples or complexes in different conformational states, often called single-particle analysis (SPA). In SPA, images of individual particles are extracted and classified representing different orientations or conformations. Advanced computational algorithms are then used to reconstruct high-resolution 3D models from these 2D images.

Cryo-EM encompasses several related techniques, including cryo-electron tomography (cryo-ET), where a series of 2D images are collected from different angles to reconstruct a 3D image of the sample, providing insights into the organization of molecular complexes within their cellular context.

The resolution revolution in cryo-EM, driven by advances in detector technology, image processing software, and electron optics, has made it a pivotal tool in structural biology. It has facilitated major breakthroughs in understanding the structure and function of complex biological machines, such as ribosomes, membrane proteins, and viruses, which are crucial for drug discovery and biotechnology.

Cryo-EM Applications

The advent of cryo-EM has had a profound impact on many fields of science. Here are some of the areas where it has made significant contributions:

• Structural Biology: Cryo-EM has enabled the determination of structures of complex biological assemblies that were previously inaccessible. These include ribosomes, virus capsids, and membrane proteins. Understanding these structures is critical for deciphering how biological molecules interact and perform their functions.
• Drug Discovery: By revealing the detailed structures of drug targets, cryo-EM aids in the rational design of new therapeutics. It allows scientists to see how potential drugs interact with their targets. This can help to develop more effective and specific treatments.
• Virology: Cryo-EM has been instrumental in the study of viruses. It has provided detailed structural information on various viruses. Examples of these viruses include the Zika virus and the SARS-CoV-2 virus responsible for COVID-19. This information is crucial for vaccine development and antiviral drug design.
• Neuroscience: Cryo-EM is being used to study the structure of proteins involved in neurological diseases, such as Alzheimer’s and Parkinson’s disease. Understanding these structures creates new avenues for therapeutic intervention.

Challenges and Future Directions

Despite its advantages, cryo-EM has limitations, including the need for expensive and sophisticated equipment, extensive computational resources for data processing, and the challenge of preparing high-quality vitrified samples. There also exist specific trade-offs between key parameters such as resolution and throughput.

Nonetheless, the continued development of cryo-EM technology and methodologies holds the promise for even greater contributions to our understanding of the molecular underpinnings of life.

Here at HWI, we are dedicated to helping you get state-of-the-art cryo-EM tools that can help you make a high resolution structural determination. It is not enough just to think about these types of high resolution 3D molecules. Instead, it is important that you see them for yourself.

By using our tools for cryo-EM, structures of molecules and viruses and protein complexes will be presented to you in the highest detail. With that said, we invite you to explore our website and see how our cryo-EM microscopy services can be applied to your research.

References & Further Reading

1. Milne JL, Borgnia MJ, Bartesaghi A, Tran EE, Earl LA, Schauder DM, Lengyel J, Pierson J, Patwardhan A, Subramaniam S. Cryo-electron microscopy–a primer for the non-microscopist. FEBS J. 2013 Jan;280(1):28-45. doi: 10.1111/febs.12078. Epub 2012 Dec 17. PMID: 23181775; PMCID: PMC3537914.
2. Callaway E. Revolutionary cryo-EM is taking over structural biology. Nature. 2020 Feb. https://www.nature.com/articles/d41586-020-00341-9. [Date Accessed: 14/03/2024]
3. Kühlbrandt, Werner. “Biochemistry. The resolution revolution.” Science (New York, N.Y.) vol. 343,6178 (2014): 1443-4. doi:10.1126/science.1251652
4. Nogales, E. The development of cryo-EM into a mainstream structural biology technique. Nat Methods 13, 24–27 (2016). https://doi.org/10.1038/nmeth.3694