The technology that allows us to witness the intricate dance of molecules in near-atomic detail.
Imagine being able to shrink down and step inside a human cell, witnessing the bustling activity of microscopic machines that perform the work of life. For decades, this was a fantasy, as the molecular machinery of life was too small and delicate to be seen in clear detail. Then came cryogenic electron microscopy (cryo-EM), a revolutionary technology that has flung open a new window into the nanoscale world, allowing scientists to create stunning 3D images of the very building blocks of biology 2 .
This technique, which earned its developers the 2017 Nobel Prize in Chemistry, has fundamentally changed the field of structural biology 4 . By flash-freezing biomolecules in action and probing them with electron beams, researchers can now determine the 3D structures of proteins, viruses, and cellular components with unprecedented clarity—all without the need for crystallization that hobbled previous methods 6 . This article explores the breathtaking advances in cryo-EM that are reshaping our understanding of life's inner workings.
Cryogenic electron microscopy is a powerful imaging technique used to determine the three-dimensional structures of biological molecules at near-atomic resolution 6 . At its core, cryo-EM involves studying samples at cryogenic temperatures (typically around -180°C) using a transmission electron microscope 1 .
The power of cryo-EM lies in its ability to image biological specimens in their native, frozen-hydrated state, preserving their natural structure in a glass-like layer of vitreous ice 4 5 . This "glass-like" ice is crucial—it forms when water is cooled so rapidly that ice crystals don't have time to develop, which would otherwise disrupt the delicate biological structures 6 .
For many years, X-ray crystallography was the dominant technique for determining protein structures. However, it required growing high-quality crystals of biological molecules—a process that could take months or years and was impossible for many complex molecular machines 4 .
Cryo-EM bypasses this limitation entirely. The dramatic improvements in cryo-EM technology over the past decade—particularly in detector technology and software algorithms—have been so transformative that scientists call it the "resolution revolution" in structural biology 4 . These advances allowed researchers to finally see biological structures in sharp detail, earning Jacques Dubochet, Joachim Frank, and Richard Henderson the 2017 Nobel Prize in Chemistry for developing the method 4 .
Jacques Dubochet, Joachim Frank, and Richard Henderson were awarded the Nobel Prize in Chemistry for developing cryo-EM.
Advances in detector technology and software algorithms have dramatically improved resolution capabilities.
The cryo-EM workflow is an elegant dance of physics, chemistry, and computational power that transforms a protein solution into a detailed 3D model.
The process begins with purifying the biological sample, which is then applied to a tiny grid. Instead of slow freezing, which would form destructive ice crystals, the grid is rapidly plunged into liquid ethane cooled by liquid nitrogen. This "plunge freezing" happens so quickly that the water solidifies into a glass-like (vitreous) state, trapping the molecules in their natural configuration 2 6 .
The frozen grid is transferred to the electron microscope, where a beam of electrons passes through the sample. As electrons hit the molecules, they scatter, casting two-dimensional shadow images onto a detector 2 . Because the molecules are frozen in random orientations, each image captures a different view of the same structure.
This is where computational magic happens. Sophisticated software algorithms analyze thousands of these 2D images, grouping similar views together and combining them to produce a detailed three-dimensional reconstruction of the molecule 2 . Researchers can then rotate this digital model in a computer, examining it from every angle to understand its form and function.
| Feature | X-ray Crystallography | NMR Spectroscopy | Cryo-EM |
|---|---|---|---|
| Sample Requirement | Large, well-ordered crystals | Soluble, small proteins | Purified solution of complexes |
| Sample State | Crystal lattice | Solution near-native state | Frozen-hydrated near-native state |
| Size Range | Small to very large complexes | Smaller proteins (< 100 kDa) | Large complexes to small proteins |
| Resolution Range | Typically very high (Å) | Lower resolution | Near-atomic to atomic (Å) |
Among the various cryo-EM techniques, Single Particle Analysis (SPA) has been particularly transformative for studying proteins and viruses 8 . Let's walk through a typical SPA experiment that might be used to determine the structure of a viral protein, such as the coronavirus spike protein.
Researchers first purify the spike protein to homogeneity, ensuring a concentration greater than 1 mg/ml in a low-salt buffer . A 3-5 microliter droplet of this solution is applied to a microscopic grid coated with a perforated carbon film .
The grid is blotted with filter paper to create a thin film of the sample, which is then rapidly plunged into liquid ethane cooled by liquid nitrogen. This instantly freezes the water into a glassy state, embedding the spike proteins in a thin layer of vitreous ice .
The grid is loaded into a cryo-electron microscope maintained at liquid nitrogen temperatures. Using an automated process, the microscope collects thousands of "movie" images—each actually a series of frames—at different angles and defocus values to compensate for the Contrast Transfer Function, an inherent property of electron microscopy that must be computationally corrected .
This computationally intensive stage involves several steps:
Researchers fit an atomic model into the final 3D electron density map, refining it to accurately represent the experimental data. The model undergoes rigorous validation before being deposited in public databases for the scientific community 5 .
The results of such an experiment can be profound. For example, determining the structure of a coronavirus spike protein reveals the exact atomic details of how the virus binds to human cells 2 . This information is crucially important for understanding infection mechanisms and designing targeted vaccines and therapeutics.
The ability to capture such structures in different states—for instance, during cell binding or membrane fusion—provides unprecedented insights into dynamic biological processes that were previously invisible to researchers 7 .
| Year | Achievement | Significance |
|---|---|---|
| 2015 | Nature Methods "Method of the Year" 4 | Recognition of cryo-EM as a transformative technology |
| 2017 | Nobel Prize in Chemistry 4 | Honor for developing cryo-EM into a powerful tool |
| 2018 | Structure of hemoglobin (64 kDa) 4 | Demonstrated application to relatively small proteins |
| 2020 | Resolution record of 1.22 Å 4 | Near-atomic resolution rivaling X-ray crystallography |
Conducting cutting-edge cryo-EM research requires specialized equipment and materials. Here are some of the essential components of a cryo-EM laboratory:
Rapidly vitrifies samples by plunging into cryogen
Sample preparationSupport film with regular perforations to hold sample
Sample preparationCryogen for rapid heat transfer during freezing
Sample preparationHigh-sensitivity camera for capturing electron images
Data acquisitionSpecialized holders for grids during automated imaging
Data acquisitionStandardized containers for cryogenic grid storage
Sample storage and managementThe evolution of cryo-EM continues at a rapid pace. The latest instruments, such as the Krios G4 installed at institutions like UCLA, offer nearly double the resolution and nine times the speed in acquiring image data compared to earlier models 7 . This means researchers can solve more structures in less time, accelerating the pace of discovery in fields ranging from cancer research to neurodegenerative diseases 7 .
Meanwhile, cryo-Electron Tomography (cryo-ET) is pushing the boundaries even further by allowing scientists to create 3D images of molecular complexes inside intact cells, providing context about how these machines operate in their native environments 8 .
When combined with artificial intelligence and machine learning approaches, these technologies promise to unlock even deeper mysteries of biological function 7 .
As these tools become more accessible and powerful, we stand at the threshold of a new era in biological understanding—one where we can not only see life's molecular machinery in stunning detail but watch it in action, leading to breakthroughs in medicine, biotechnology, and our fundamental comprehension of life itself.