Cryomicroscopy, particularly cryogenic electron microscopy (cryo-EM), visualizes biological molecules and cellular structures in a state closely resembling their natural environment. This method captures high-resolution, three-dimensional images of components like proteins, viruses, and cellular machinery at a near-atomic level of detail. The development of cryomicroscopy has transformed structural biology, providing significant insights into the architecture and function of life’s fundamental building blocks.
The Need for Cryomicroscopy
For many years, scientists relied on traditional microscopy techniques like standard electron microscopy or X-ray crystallography to study biological structures. These methods often limited studies of delicate biological samples. Traditional electron microscopy requires samples to be stained, dehydrated, or placed in a vacuum, which can alter or damage their native forms. The high-energy electron beams used can also damage sensitive biological material.
X-ray crystallography, while capable of high resolution, requires biological molecules to be crystallized. Many important proteins and molecular complexes are challenging or impossible to crystallize, leaving their structures unknown. These techniques often failed to provide an accurate picture of biological components as they exist and function within living systems. A new approach was needed to overcome these obstacles and observe biological structures in a more preserved state, free from conventional preparation artifacts.
How Cryomicroscopy Works
Cryomicroscopy addresses the challenges of traditional methods by freezing samples so rapidly that damaging ice crystals do not form. This process, known as vitrification, involves flash-freezing the biological sample in a thin layer of water. A purified sample solution is applied to a specialized grid, blotted to create a thin film, and then plunged into a cryogen like liquid ethane, cooled by liquid nitrogen to temperatures below -150 °C, often around -188 °C. This transforms the water into an amorphous, glass-like solid, preserving molecules in their native, hydrated state.
Once vitrified, samples are maintained at low temperatures within the electron microscope. Imaging at cryogenic temperatures reduces the radiation damage caused by the electron beam, allowing for clearer, more detailed images. The electron microscope then captures multiple two-dimensional (2D) images of the sample from various angles. As individual molecules in the vitrified ice are frozen in random orientations, each 2D image provides a different view.
Computational algorithms then process these 2D images. Similar projections are grouped, and software combines them to reconstruct a high-resolution, three-dimensional (3D) model of the molecule. This 3D reconstruction reveals the precise architecture of the biological structure, providing insights into its function that were previously unattainable. The process refines orientation assignments from the 2D images, leading to increasingly accurate and detailed 3D maps.
Applications of Cryomicroscopy
Cryomicroscopy has provided views into a wide array of biological structures and processes, clarifying their functions. It has been impactful in virology, revealing detailed structures of many viruses. For instance, cryo-EM helped determine the structure of the Zika virus at resolutions as fine as 3.1 Å, providing insights into its protein interactions and potential drug-binding sites. Researchers have also used cryo-EM to image the Ebola virus protein, contributing to an understanding of its replication and infection mechanisms.
During the COVID-19 pandemic, cryo-EM elucidated the structure of the SARS-CoV-2 spike protein, a key component for the virus’s entry into human cells. This rapid structural determination accelerated vaccine and treatment development efforts. Beyond viruses, cryomicroscopy has provided detailed structures of large protein complexes, membrane proteins, and cellular machinery.
In neurodegenerative diseases, cryomicroscopy has provided insights into protein aggregates associated with conditions like Alzheimer’s and Parkinson’s diseases. It has resolved the structures of tau filaments from Alzheimer’s patients. Understanding these detailed structures can inform the design of targeted therapies.
Impact on Scientific Discovery
Cryomicroscopy has significantly shifted structural biology and related fields. Its ability to visualize biomolecules in a near-native state has deepened our understanding of life at the molecular level. This advancement has accelerated fundamental biological research, allowing scientists to explore complex molecular mechanisms underlying health and disease with unprecedented clarity.
Cryomicroscopy is a tool in drug discovery and vaccine development. By mapping the structures of disease-related proteins and viral components, it enables the design of new therapeutic compounds and vaccines. Understanding the structure of viral surface proteins, for example, aids in developing effective vaccine candidates and antiviral drugs. This technology helps identify potential drug-binding targets and optimize drug candidates.
The impact of cryo-EM was recognized with the 2017 Nobel Prize in Chemistry, awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson. Their contributions to developing and refining the technique transformed biochemistry, making atomic-resolution images of biomolecules routinely achievable. This capability continues to drive scientific breakthroughs and holds potential for future advancements in medicine and biotechnology.