Cryogenic electron microscopy

Cryogenic electron microscopy (cryo-EM) is a transmission electron microscopy technique applied to samples cooled to cryogenic temperatures. Developed in the 1970s, advances in detector technology and software allow biomolecular structures to be imaged at near-atomic resolution. The approach has become a popular alternative to X-ray crystallography or NMR spectroscopy in structural biology.

When scanning biological specimens, sample structure is preserved by embedding in vitreous ice. An aqueous sample solution is applied to a grid-mesh and plunge-frozen in liquid ethane or a mixture of liquid ethane and propane.

The 2017 Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution." Nature Methods also named cryo-EM as the "Method of the Year" in 2015.

History

Early development

In the 1960s, transmission electron microscopy of biological samples was limited because of radiation damage from the high energy electron beams. Scientists hypothesized that examining specimens at low temperatures would reduce beam-induced radiation damage. Both liquid helium (−269 °C or 4 K or −452 °F) and liquid nitrogen (−195.79 °C or 77 K or −320 °F) were considered as cryogens, however high stability was never achieved. In 1980, Erwin Knapek and Jacques Dubochet published comments on beam damage at cryogenic temperatures sharing observations that:

Thin crystals mounted on carbon film were found to be from 30 to 300 times more beam-resistant at 4 K than at room temperature... Most of our results can be explained by assuming that cryoprotection in the region of 4 K is strongly dependent on the temperature.

However, these results were not reproduced and just two years later amendments were published, along with a commentary in Nature, indicating that the beam resistance was less significant than anticipated. The protection gained at 4 K was closer to "tenfold for standard samples of L-valine", than what was previously stated. While cryo-EM samples are routinely collected at liquid nitrogen temperatures, work has continued to understand sample behavior at liquid helium temperatures.

In 1981 scientists at the European Molecular Biology Laboratory, reported the first successful cryo-EM. Researchers used a thin film of vitrified pure water a hydrophilic carbon film that was rapidly plunged into cryogen (liquid propane or liquid ethane cooled to 77 K). The thin layer of amorphous ice was less than 1 μm thick and an electron diffraction pattern confirmed the presence of amorphous/vitreous ice. In 1984 the group demonstrated the power of cryo-EM in structural biology by analysing vitrified adenovirus type 2, T4 bacteriophage, Semliki Forest virus, Bacteriophage CbK, and Vesicular-Stomatitis-Virus. The paper marked the origin of Cryo-EM, and the technique has become routine in laboratories throughout the world.

The energy of the electrons used for imaging (80–300 kV) can break covalent bonds in organic and biological samples. Imaging biological specimens requires minimising electron exposure. Low exposures require images of thousands or millions of frozen molecules be selected, aligned, and averaged to obtain high-resolution maps, using specialized software. The 2012 introduction of direct electron detectors and better computational algorithms significantly improved structural features.

Recent advancements

Direct Electron Detectors, and more powerful imaging algorithms allow macromolecular structures to be determined at near-atomic resolution. Imaged macromolecules include viruses, ribosomes, mitochondria, ion channels, and enzyme complexes. Starting in 2018, cryo-EM could be applied to structures as small as hemoglobin (64 kDa) with resolutions up to 1.8 Å. In 2019, cryo-EM structures grew to 2.5% of structures deposited in the Protein Data Bank. Cryo-EM can be used for cryo-electron tomography (cryo-ET), creating 3D reconstructions of samples from tilted 2D images.

The 2010s saw drastic advancements of electron cameras, including direct electron detectors, causing a "resolution revolution" pushing the resolution barrier beneath the crucial ~2-3 Å limit to resolve amino acid position and orientation.

Richard Henderson (MRC Laboratory of Molecular Biology, Cambridge, UK) formed a consortium with engineers at the Rutherford Appleton Laboratory and scientists at the Max Planck Society to fund and develop a first prototype. The consortium then joined forces with the electron microscope manufacturer FEI to roll out and market the new design. At about the same time, Gatan Inc. of Pleasanton, California came out with a similar detector designed by Peter Denes (Lawrence Berkeley National Laboratory) and David Agard (University of California, San Francisco). A third type of camera was developed by Nguyen-Huu Xuong at the Direct Electron company (San Diego, California).

Recent advancements in protein-based imaging scaffolds assist with sample orientation bias and size limit. Though the minimum size for Cryo-EM remains undetermined, proteins smaller than ~50 kDa generally have too low a signal-to-noise ratio (SNR) to resolve protein particles, making 3D reconstruction difficult or impossible. Multiple techniques have been reported to improve SNR when determining the structures of small proteins. Based on high-affinity DARPins, nanobodies, antibody fragments, these methods rigidly bind the target protein and thereby increase the effective particle size and introduce symmetry to improve SNR for Cryo-EM map reconstruction. An advantage of Cryo-EM over crystallization is that it requires much less sample material. This makes it easier to determine structures of proteins that cannot be isolated with high yield.

2017 Nobel Prize in Chemistry

In recognition of the impact cryo-EM has had on biochemistry, three scientists, Jacques Dubochet, Joachim Frank and Richard Henderson, were awarded the Nobel Prize in Chemistry "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution."

Techniques

Mode of electron microscopy

Cryogenic transmission electron microscopy

Cryogenic transmission electron microscopy (cryo-TEM) is a transmission electron microscopy technique that is used in structural biology and materials science. Colloquially, the term "cryogenic electron microscopy" or its shortening "cryo-EM" refers to cryogenic transmission electron microscopy by default, as the vast majority of cryo-EM is done in transmission electron microscopes, rather than scanning electron microscopes.

Correlative light cryo-TEM and cryo-ET

In 2019, correlative light cryo-TEM and cryo-ET were used to observe tunnelling nanotubes (TNTs) in neuronal cells.

Scanning electron cryomicroscopy

Scanning electron cryomicroscopy (cryoSEM) is a scanning electron microscopy technique with a scanning electron microscope's cold stage in a cryogenic chamber.

Technique of use and data analysis

Electron cryotomography

In electron cryotomography (cyro-ET), many pictures of a sample are taken from different angles using a tilting mechanism. The images are combined to create a 3D model (map) of ~1–4 nm resolution.

Single particle analysis

SPA or single-particle cyro-EM is the method used to obtain near-atomic resolution (<1 nm) models of biomolecules. It is what the 2017 Nobel Prize refers to. In SPA, a large collection of cyro-TEM images are automatically sorted into classes. Within each class, the images are combined to reduce noise and to create a 3D model of the class of particles, a 3D "map". The main innovation compared to cyro-ET is the combination of images from similar objects.

When combined with a knowledge of time progression, the result is time-resolved cyro-TEM.

Comparisons to X-ray crystallography

Traditionally, X-ray crystallography has been the most popular technique for determining the 3D structures of biological molecules. However, the aforementioned improvements in cryo-EM have increased its popularity as a tool for examining the details of biological molecules. Since 2010, yearly cryo-EM structure deposits have outpaced X-ray crystallography. Though X-ray crystallography has drastically more total deposits due to a decades-longer history, total deposits of the two methods are projected to eclipse around 2035.

The resolution of X-ray crystallography is limited by crystal homogeneity, and coaxing biological molecules with unknown ideal crystallization conditions into a crystalline state can be very time-consuming, in extreme cases taking months or even years. To contrast, sample preparation in cryo-EM may require several rounds of screening and optimization to overcome issues such as protein aggregation and preferred orientations, but it does not require the sample to form a crystal, rather samples for cryo-EM are flash-frozen and examined in their near-native states.