A Beginner's Guide to Cryo-Electron Microscopy (Cryo-EM)
Structural biology has undergone a seismic shift with the rise of cryogenic electron microscopy (cryo-EM). Once a niche technique, cryo-EM is now at the forefront of molecular and cellular imaging, providing researchers with the ability to visualize biological structures at near-atomic resolution. But what exactly is cryo-EM, and why has it become such a pivotal tool in modern science? This guide will take you through the basics of cryo-EM, its key methodologies, and its transformative impact to the fields of structural biology.
What is Cryo-EM?
Cryo-electron microscopy, or cryo-EM, is a form of electron microscopy where samples are studied at cryogenic temperatures (below -150°C). Unlike traditional methods that may require extensive sample preparation, including staining or crystallization, cryo-EM allows researchers to observe biological specimens in their near-native state, frozen in a thin layer of vitreous ice. This technique has revolutionized our ability to study complex biological molecules, viruses, and cellular components in unprecedented detail.
Transmission Electron Microscopes, the backbone of cryo-EM, use electrons instead of light. The interaction between the electrons and the specimen yields high resolution images, which can be further analyzed to extract structural information.
The Importance of Cryo-EM in Structural Biology
Cryo-EM's rise to prominence can be traced back to its ability to fill a crucial gap in structural biology. While X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have long been the go-to methods for determining molecular structures, they come with limitations. X-ray crystallography requires well-ordered crystals, which are often difficult or impossible to obtain for large or flexible molecules. NMR, while powerful, is best suited for smaller proteins but not for large macromolecular complexes.
Cryo-EM, on the other hand, is routinely used to solve large and complex structures that are challenging for other techniques. It has proven particularly valuable in resolving the structures of macromolecular assemblies, membrane proteins, and even entire viruses. The ability to visualize these structures in their near-native environments has provided insights that were previously out of reach.
A Brief History of Cryo-EM
The story of cryo-EM began in 1933 when Max Knoll and Ernst Ruska developed the first transmission electron microscope. This breakthrough was a major milestone in microscopy, enabling scientists to observe particles much smaller than what was possible with optical microscopes. It laid the groundwork for future developments in electron microscopy, including the cryogenic techniques that would come decades later.
In the 1970s, scientists faced a significant challenge: how to minimize radiation damage to biological samples during electron microscopy. The solution emerged from cryogenic techniques, which examined samples at extremely low temperatures. This approach, validated using two-dimensional or thin three-dimensional crystals showed a substantial reduction in radiation damage out to high resolution at temperatures close to liquid nitrogen or even liquid helium. In the same decade, Joachim Frank pioneered the development of image processing techniques that would become essential to cryo-EM. Frank’s work focused on transforming the often noisy and blurry 2D images captured by electron microscopes into clear, interpretable 3D structures. This ability to extract meaningful structural information from cryo-EM data was a game-changer for the field, allowing scientists to visualize complex biological molecules in unprecedented detail.
A stumbling block to studying biological systems in their native state is the presence of water and the requirement for the electron microscope to be kept at high vacuum. Parsons was able to circumvent this using a wet cell, or environmental chamber, in the microscope employing a set of membranes to keep the protein in water whilst avoiding the protein-destroying boil-off that the vacuum would cause. Currently, a large body of work exists using this very principle to study in-situ events using special purpose specimen holders. Yet, the large amount of water in a wet cell would result in substantial loss of signal and the radiation damage caused at room temperature meant that this technique would not result in high-resolution structures. A major leap forward occurred in 1981 when Jacques Dubochet and Alasdair McDowall introduced the method of rapid freezing, or vitrification. Thus, a specimen carrier with a small amount of water was quenched in liquid ethane kept close to its melting point using liquid nitrogen. The rapid cool down, occurring at a rate of roughly 105K/sec, would ensure crystalline water would not form and the sample would be suspended in a thin layer of vitreous ice. This innovation was crucial in protecting specimens from dehydration and damage from ice crystals enabling researchers to observe them in a native state.
Cryo-EM's full potential became evident in 1990 when Richard Henderson and his colleagues determined the first atomic resolution structure of a biomacromolecule—bacteriorhodopsin—using cryo-EM applied to two-dimensional crystals. The entire amino acid sequence could be folded into the three-dimensional map of the protein. This achievement demonstrated that cryo-EM could produce atomic-level details of biological structures, opening new possibilities for research in structural biology.
A transformative leap in cryo-EM was made in 2012-2013 with the introduction of direct electron detectors. These new detectors captured images using a movie mode, meaning that the primary obstacle in cryo-EM, beam-induced motion, could be largely corrected by aligning the individual frames in the movie. This breakthrough led to significantly improved image quality and resolution, resulting in what is colloquially known as the "resolution-revolution" in cryo-EM. Along with improved automation and reliability of the electron microscopes, this development marked the technique's transition into a highly reliable and widely adopted method for determining the structures of complex biomolecules.
The importance of cryo-EM was globally recognized in 2017 when the Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson. Their pioneering contributions to the development of cryo-EM were acknowledged as a breakthrough that had fundamentally changed the field of structural biology.
How Cryo-EM Works
The process of cryo-EM can be broken down into several key steps:
- Sample Preparation: The first and most critical step in cryo-EM is the preparation of the sample. A purified sample is applied to a grid, and excess liquid is blotted away, leaving a thin film of the sample. This grid is then rapidly plunged into liquid ethane, freezing the sample at a rate that prevents ice crystal formation and preserves the sample in a vitreous state. Many variations exist on this topic ranging from the type of carrier film used, such as carbon, graphene (derivatized or not) or gold foils for instance, to the method of applying the sample, applying either a droplet or dispensing the sample onto a grid using a stylus or an inkjet technique to name a few. Carrier film with holes at regular intervals are routinely used at this stage.
- Data Collection: Once the sample is vitrified, it is loaded into a transmission electron microscope operating at cryogenic temperatures. Often, many grids of the same sample are loaded as freezing can still produce variation from sample to sample meaning subtly different imaging conditions are needed. Specific holes as identified from low-magnification images are targeted at higher magnification after which multiple images are acquired with a direct detector using beam shift plus image shift techniques in order to minimize the time required for the stage to settle. Typically, thousands of images and millions of particle images are needed for a high-resolution structure, even more so if the sample exhibits conformational flexibility.
- Image Processing: The captured images are subjected to a process called motion correction, where individual frames of the images are aligned to account for any movement of the sample during imaging. Next, the effects of defocus on each image is analyzed in a process called CTF estimation. Particles are then selected and extracted from the micrographs as 2D images, and sorted based on similarity in a process called 2D classification. Finally, 2D classes are chosen and used to reconstruct a final 3D model, which can be further refined to improve resolution and used to fit in amino acid chains and build a structure.
Applications and Future Directions
As a powerful and versatile tool in structural biology, cryo-EM provides researchers with unparalleled insights into the molecular architecture of biological systems. Its broad range of applications and the promising future directions for this technology continue to drive advancements in various fields.
Structure Determination of Biomolecules
Cryo-EM has become the method of choice for determining the structures of large and complex biomolecules at near-atomic resolution. This technique excels in visualizing macromolecular assemblies that are difficult to study using traditional methods like X-ray crystallography or NMR spectroscopy. Notable examples of structures elucidated by cryo-EM include protein complexes, viruses, ribosomes, membrane proteins, and spliceosomes.
New methods are being developed to extend cryo-EM’s reach to more challenging and diverse samples:
- Membrane Proteins and Lipid-Protein Complexes: Techniques are being refined to better study these difficult targets, which are vital for understanding cellular processes and drug interactions.
- Intrinsically Disordered Proteins: Cryo-EM is evolving to capture the structures of these flexible and dynamic proteins, which play critical roles in cell signaling and regulation.
- Large Macromolecular Assemblies: As cryo-EM technology improves, it will become increasingly feasible to study very large complexes, shedding light on their intricate workings.
Drug Discovery and Development
The pharmaceutical industry has increasingly adopted cryo-EM as a key tool in drug discovery and development. Its ability to rapidly provide high-resolution structures of drug targets enables the design of more effective therapeutics. The atomic structures of complexes provided by cryo-EM also open the door to rational drug design using powerful docking tools of ligands, cofactors or effector molecules into active sites. Applications in this area include structure-based drug design, fragment-based drug discovery, PROTACs development, and antibody drug development.
The integration of cryo-EM into drug discovery is expected to grow, with several promising applications:
- High-Throughput Screening: Cryo-EM could be used for the rapid screening of drug candidates, accelerating the early stages of drug development.
- Studying Drug-Target Interactions: The ability to visualize these interactions at atomic resolution will provide insights that are crucial for designing more effective drugs.
- Investigating Drug Resistance: Cryo-EM will help researchers understand the structural basis of drug resistance, guiding the development of next-generation therapeutics.
Cellular and Molecular Biology
Cryo-EM’s ability to capture the detailed architecture of cellular components has made it an invaluable tool in cellular and molecular biology. Key applications include studying protein-protein interactions, examining conformational changes, and investigating cellular organelles.
Advancing cryo-EM techniques to study biomolecules within their native cellular environments will open new frontiers in structural biology:
- Cryo-Electron Tomography: This approach will allow for the 3D visualization of intact cells, providing insights into the spatial organization and interactions of cellular components.
- Correlative Light and Electron Microscopy: Combining cryo-EM with light microscopy will enable the study of dynamic processes within cells, bridging the gap between molecular structures and cellular functions.
Closing Thoughts
Cryo-EM has firmly established itself as a powerhouse in structural biology. Its ability to provide detailed images of biological structures at near-atomic resolution has opened new frontiers in our understanding of molecular and cellular processes. As the technology continues to evolve, cryo-EM is set to play an even more central role in future scientific discoveries. Whether you're a seasoned researcher or a curious beginner, the world of cryo-EM offers endless opportunities for exploration and discovery.
References & Further Reading:
- Savva C. A beginner's guide to cryogenic electron microscopy. Biochem (Lond). 2019;41 (2):46–52. doi:10.1042/BIO04102046.
- Cheng Y. Single-particle cryo-EM-How did it get here and where will it go. Science. 2018;361(6405):876-880. doi:10.1126/science.aat4346.
- Benjin X, Ling L. Developments, applications, and prospects of cryo-electron microscopy. Protein Sci. 2020;29(4):872-882. doi:10.1002/pro.3805.
- Du YM, Gao YZ, Jia XD, et al. Applications and prospects of cryo-EM in drug discovery. Military Medical Research. 2023;10(1):10. doi:10.1186/s40779-023-00446-y.