Cryogenic electron microscopy
Cryogenic electron microscopy is a transmission electron microscopy technique applied to samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of 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. While development of the technique began in the 1970s, recent advances in detector technology and software algorithms have allowed for the determination of biomolecular structures at near-atomic resolution. This has attracted wide attention to the approach as an alternative to X-ray crystallography or NMR spectroscopy in the structural biology field.
In 2017, the 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, the use of transmission electron microscopy for structure determination of biological samples was limited because of the radiation damage due to high energy electron beams. Scientists hypothesized that examining specimens at low temperatures would reduce beam-induced radiation damage. Both liquid helium and liquid nitrogen 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 reproducible and just two years later amendments were published, along with a commentary in Nature, indicating that the beam resistance was less significant than initially 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 and collect at liquid helium temperatures.
In 1981, Alasdair McDowall and Jacques Dubochet, scientists at the European Molecular Biology Laboratory, reported the first successful implementation of cryo-EM. McDowall and Dubochet vitrified pure water in a thin film by spraying it onto a hydrophilic carbon film that was rapidly plunged into cryogen. 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, Dubochet's group demonstrated the power of cryo-EM in structural biology with analysis of vitrified adenovirus type 2, T4 bacteriophage, Semliki Forest virus, Bacteriophage CbK, and Vesicular-Stomatitis-Virus. This paper is generally considered to mark the origin of Cryo-EM, and the technique has been developed to the point of becoming routine at numerous laboratories throughout the world.
The energy of the electrons used for imaging can lead to the breaking of covalent bonds in organic and biological samples. When imaging such specimens it is necessary to limit the electron exposure used to acquire the image. These low exposures require that the images of thousands or even millions of identical frozen molecules be selected, aligned, and averaged to obtain high-resolution maps, using specialized software. A significant improvement in structural features was achieved in 2012 by the introduction of direct electron detectors and better computational algorithms.
Recent advancements
Advances in electron detector technology, particularly Direct Electron Detectors, as well as more powerful software imaging algorithms have allowed for the determination of macromolecular structures 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 and with resolutions up to 1.8 Å. In 2019, cryo-EM structures represented 2.5% of structures deposited in the Protein Data Bank, and this number continues to grow. An application of cryo-EM is cryo-electron tomography, where a 3D reconstruction of the sample is created from tilted 2D images.The 2010s were marked with drastic advancements of electron cameras. Notably, the improvements made to direct electron detectors have led to a "resolution revolution" pushing the resolution barrier beneath the crucial ~2-3 Å limit to resolve amino acid position and orientation.
Henderson 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 and David Agard. A third type of camera was developed by Nguyen-Huu Xuong at the Direct Electron company.
More recently, advancements in the use of protein-based imaging scaffolds are helping to solve the problems of 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 to be able to resolve protein particles in the image, 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.
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
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 in neuronal cells.Scanning electron cryomicroscopy
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, many pictures of a sample are taken from different angles using a tilting mechanism. The images are combined to create a 3D model of ~1–4 nm resolution.Single particle analysis
SPA or single-particle cyro-EM is the method used to obtain near-atomic resolution 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.
According to Proteopedia, the median resolution achieved by X-ray crystallography on the Protein Data Bank is 2.05 Å, and the highest resolution achieved on record is 0.48 Å. As of 2020, the majority of the protein structures determined by cryo-EM are at a lower resolution of 3–4 Å. However, as of 2020, the best cryo-EM resolution has been recorded at 1.22 Å, making it a competitor in resolution in some cases.
Electron crystallography
Similar to X-ray crystallography used to determine the crystal structure of molecules of different sizes using the X-ray diffration pattern, electrons can also produce a electron diffraction pattern from a crystal.Cyro-EC is typically done with 3D crystals, but it has also been used in analysis of two-dimensional crystals and nalysis of helical filaments or tubes.
Microcrystal electron diffraction is a version of electron crystallography that works with crystals a billion times smaller than what X-ray diffraction requires, which is advantagous when very little crystal is available. It has been extensively used to determine the structure of large biomolecules. It is also very useful in studying small molecules, from peptides to simpler compounds.