Cryo-electron microscopy

Cryo-electron microscopy (cryo-EM) is a form of transmission electron microscopy (TEM) where the sample is studied at cryogenic temperatures. It has become an essential technique in structural biology for studying the three-dimensional structures of biomolecules. By rapidly freezing the samples, cryo-EM preserves their native state, allowing for the observation of structures that are difficult or impossible to crystallize. This method has revolutionized the field of structural biology, providing insights into the architecture of large biological macromolecules and complexes.

Overview

Cryo-EM involves the rapid cooling of samples to cryogenic temperatures without the formation of ice crystals, which could damage the sample structure. This process, known as vitrification, is achieved by plunging the samples into liquid ethane cooled by liquid nitrogen. The vitrified samples are then examined in a TEM equipped with a cryo-stage, which keeps the samples at low temperatures during observation.

The technique has several modalities, with single-particle analysis (SPA) being the most widely used. SPA allows for the reconstruction of high-resolution three-dimensional structures from two-dimensional images of randomly oriented particles. Other modalities include cryo-electron tomography, where a series of images are taken at different angles to reconstruct the 3D structure of the sample, and electron crystallography, used for studying well-ordered arrays of molecules.

Applications

Cryo-EM has been instrumental in elucidating the structures of a wide range of biological molecules, including proteins, viruses, and large molecular complexes. Its ability to image molecules in their native state, without the need for crystallization, makes it particularly valuable for studying membrane proteins, flexible proteins, and large complexes that are challenging to crystallize. Cryo-EM has contributed to significant scientific discoveries, such as the structure of the ribosome, the Zika virus, and the spike protein of the SARS-CoV-2 virus, providing critical insights into their functions and mechanisms.

Advantages and Limitations

The primary advantage of cryo-EM is its ability to visualize biological macromolecules in a state that closely resembles their natural environment. Unlike X-ray crystallography, it does not require crystallization, enabling the study of molecules that are difficult or impossible to crystallize. Cryo-EM can also study large complexes in their entirety, providing a comprehensive view of their structure and function.

However, cryo-EM has limitations. High-resolution structure determination requires a large number of images and sophisticated computational methods for image processing and 3D reconstruction. The technique is also limited by the sensitivity and resolution of the electron microscope and detectors.

Recent Developments

Recent advances in electron detector technology and image processing software have significantly improved the resolution achievable with cryo-EM, with some structures now being resolved at near-atomic resolution. These developments have expanded the range of biological questions that can be addressed using cryo-EM and have cemented its role as a cornerstone technique in structural biology.

Conclusion

Cryo-electron microscopy has emerged as a powerful tool for understanding the molecular architecture of life. By providing detailed images of biological molecules in their native state, it offers unparalleled insights into the workings of the cell at the molecular level. As technology and methods continue to evolve, cryo-EM is poised to make even more significant contributions to our understanding of biological systems.