cryo-electron microscopy

cryo-electron microscopy
Name	Cryo-electron microscopy
Acronym	Cryo-EM
Classification	Electron microscopy
Inventor	Jacques Dubochet, Joachim Frank, Richard Henderson
Year	1980s onward
Related	Single-particle analysis, Tomography (imaging), X-ray crystallography

cryo-electron microscopy. It is a form of transmission electron microscopy where biological samples are rapidly frozen to preserve their native structure in a thin layer of vitreous ice. This technique allows for the high-resolution determination of macromolecular complexes, viruses, and cellular structures without the need for crystallization. Its development, recognized by the 2017 Nobel Prize in Chemistry, has revolutionized structural biology by enabling the visualization of previously intractable biological assemblies.

History and development

The foundational work for this method began in the 1970s and 1980s through the independent efforts of key pioneers. Richard Henderson and Nigel Unwin used electron diffraction on bacteriorhodopsin in purple membrane to demonstrate that near-atomic resolution was possible. Concurrently, Joachim Frank developed computational methods for single-particle analysis, enabling the averaging of images of identical particles. A critical breakthrough came from Jacques Dubochet and his team at the European Molecular Biology Laboratory, who perfected the technique of plunge freezing samples into liquid ethane to form amorphous ice, thus preventing damaging ice crystals. These combined innovations were formally recognized with the awarding of the Nobel Prize in Chemistry to Dubochet, Frank, and Henderson. Subsequent technological leaps, including the development of direct electron detectors by companies like Gatan, Inc. and FEI Company, and improved software from institutions like the MRC Laboratory of Molecular Biology, ushered in the "resolution revolution" in the 2010s.

Principles and technique

The core principle involves imaging a sample maintained at cryogenic temperatures, typically using liquid nitrogen, within the vacuum of a transmission electron microscope. A beam of electrons is transmitted through the vitrified sample, and interactions with the specimen produce a two-dimensional projection image. For three-dimensional reconstruction, multiple images are collected from different orientations, either by tilting the specimen in tomography or by imaging many randomly oriented copies of a purified complex in single-particle analysis. The use of a field emission gun provides a coherent electron beam, while the direct electron detector records images with high signal-to-noise ratio, capturing movies that allow for correction of beam-induced motion.

Sample preparation

Successful analysis hinges on preparing a thin, frozen-hydrated layer of the sample. The standard method is plunge freezing, where a small volume of purified protein or virus suspension is applied to a grid made of copper or gold with a holey carbon support. The grid is then rapidly blotted and plunged into a cryogen like liquid ethane cooled by liquid nitrogen. This ultra-rapid cooling, at rates exceeding 10,000 Kelvin per second, prevents the formation of crystalline ice, instead trapping the sample in a glass-like, vitreous state. For thicker samples like cells or tissues, methods such as high-pressure freezing followed by cryo-focused ion beam milling at facilities like the Janelia Research Campus are used to create thin, electron-transparent lamellae.

Data processing and analysis

The path from raw images to an atomic model is computationally intensive. For single-particle analysis, millions of particle images are automatically picked from micrographs using programs like RELION, cryoSPARC, or cisTEM. These particles undergo iterative rounds of two-dimensional classification, three-dimensional classification, and refinement to align and average them, progressively improving the resolution of the final three-dimensional reconstruction. The final density map is then interpreted by fitting known atomic models from the Protein Data Bank or building new ones *de novo* using software such as Coot or PHENIX. The quality of the map is assessed by metrics like the Fourier shell correlation and the global resolution.

Applications and impact

This methodology has had a transformative impact across biology and medicine. It has been instrumental in determining the structures of massive complexes like the ribosome, spliceosome, and nuclear pore complex. It elucidated the architecture of key membrane proteins such as the TRPV1 ion channel and G protein-coupled receptors. During the COVID-19 pandemic, researchers at institutions like the University of Texas at Austin and the University of Oxford used it to rapidly solve the structures of the SARS-CoV-2 spike protein and its complexes with neutralizing antibodies, directly informing vaccine design by Pfizer and Moderna. It is also pivotal in drug discovery for visualizing drug-target interactions.

Limitations and challenges

Despite its power, the technique faces several constraints. Sample preparation remains a major bottleneck, as achieving ideal ice thickness and particle distribution is often empirical and sample-dependent. The high cost of the electron microscope itself, often from manufacturers like Thermo Fisher Scientific or JEOL, and the required infrastructure for cryogen handling are significant barriers. Beam sensitivity of biological specimens limits the total electron dose, inherently capping the attainable resolution for some fragile targets. While automation has improved, data processing still requires substantial expertise in computational biology and access to high-performance computing resources, such as those at the National Center for Supercomputing Applications.

Category:Electron microscopy Category:Structural biology Category:Biophysical techniques