Cryogenic electron microscopy

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. Amendments were published two years later, 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. work has continued to understand sample behavior at liquid helium temperatures. Researchers sprayed pure water onto 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 formed on the film 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 to 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). 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 workflow 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. 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 (as of May 19, 2019) on the Protein Data Bank is 2.05 Å, As of 2020, the majority of the protein structures determined by cryo-EM (single particle analysis) 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, Cyro-EC is typically done with 3D crystals, but it has also been used in analysis of two-dimensional crystals and analysis of helical filaments or tubes. Microcrystal electron diffraction (MicroED) is a version of electron crystallography that works with crystals a billion times smaller than what X-ray diffraction requires. It has been used to determine the structure of large biomolecules (proteins, nucleic acids, their complexes). It is also very useful in studying small molecules, from peptides to simpler compounds. == Specimen handling for imaging ==

(This section does not apply to electron crystallography.) Biological specimens Thin film The biological material is spread on an electron microscopy grid and is preserved in a frozen-hydrated state by rapid freezing, usually in liquid ethane near liquid nitrogen temperature. By maintaining specimens at liquid nitrogen temperature or colder, they can be introduced into the high-vacuum of the electron microscope column. Most biological specimens are extremely radiosensitive, so they must be imaged with low-dose techniques (usefully, the low temperature of transmission electron cryomicroscopy provides an additional protective factor against radiation damage). Consequently, the images are extremely noisy. For some biological systems it is possible to average images to increase the signal-to-noise ratio and retrieve high-resolution information about the specimen using the technique known as single particle analysis. This approach in general requires that the things being averaged are identical, although some limited conformational heterogeneity can now be studied (e.g. ribosome). Three-dimensional reconstructions from CryoTEM images of protein complexes and viruses have been solved to sub-nanometer or near-atomic resolution, allowing new insights into the structure and biology of these large assemblies. Analysis of ordered arrays of protein, such as 2-D crystals of transmembrane proteins or helical arrays of proteins, also allows a kind of averaging which can provide high-resolution information about the specimen. This technique is called electron crystallography. Vitreous sections The thin film method is limited to thin specimens (typically < 500 nm) because the electrons cannot cross thicker samples without multiple scattering events. Thicker specimens can be vitrified by plunge freezing (cryofixation) in ethane (up to tens of μm in thickness) or more commonly by high pressure freezing (up to hundreds of μm). They can then be cut in thin sections (40 to 200 nm thick) with a diamond knife in a cryoultramicrotome at temperatures lower than −135 °C (devitrification temperature). The sections are collected on an electron microscope grid and are imaged in the same manner as specimen vitrified in thin film. This technique is called transmission electron cryomicroscopy of vitreous sections (CEMOVIS) or transmission electron cryomicroscopy of frozen-hydrated sections. Material specimens In addition to allowing vitrified biological samples to be imaged, CryoTEM can also be used to image material specimens that are too volatile in vacuum to image using standard, room temperature electron microscopy. For example, vitrified sections of liquid-solid interfaces can be extracted for analysis by CryoTEM, and sulfur, which is prone to sublimation in the vacuum of electron microscopes, can be stabilized and imaged in CryoTEM. == Image processing in cryo-TEM ==

Even though in the majority of approaches in electron microscopy one tries to get the best resolution image of the material, it is not always the case in cryo-TEM. Besides all the benefits of high resolution images, the signal to noise ratio remains the main hurdle that prevents assigning orientation to each particle. For example, in macromolecule complexes, there are several different structures that are being projected from 3D to 2D during imaging and if they are not distinguished the result of image processing will be a blur. That is why the probabilistic approaches become more powerful in this type of investigation. There are two popular approaches that are widely used nowadays in cryo-EM image processing, the maximum likelihood approach that was discovered in 1998 and relatively recently adapted Bayesian approach. The maximum likelihood estimation approach comes to this field from the statistics. Here, all the possible orientations of particles are summed up to get the resulting probability distribution. We can compare this to a typical least square estimation where particles get exact orientations per image. This way, the particles in the sample get "fuzzy" orientations after calculations, weighted by corresponding probabilities. The whole process is iterative and with each next iteration the model gets better. The good conditions for making the model that closely represent the real structure is when the data does not have too much noise and the particles do not have any preferential direction. The main downside of maximum likelihood approach is that the result depends on the initial guess and model optimization can sometimes get stuck at local minimum. The Bayesian approach that is now being used in cryo-TEM is empirical by nature. This means that the distribution of particles is based on the original dataset. Similarly, in the usual Bayesian method there is a fixed prior probability that is changed after the data is observed. The main difference from the maximum likelihood estimation lies in special reconstruction term that helps smoothing the resulting maps while also decreasing the noise during reconstruction. the program became widespread in the cryo-TEM field. It offers a range of corrections that improve the resolution of reconstructed images, allows implementing versatile scripts using python language and executes the usual tasks of 2D/3D model classifications or creating de novo models. == Gallery ==

File:Cryoem groel.jpg|Cryo-EM image of GroEL suspended in amorphous ice at × magnification File:Structure-of-Alcohol-Oxidase-from-Pichia-pastoris-by-Cryo-Electron-Microscopy-pone.0159476.s006.ogv|Structure of alcohol oxidase from Pichia pastoris by Cryo-EM File:25K15pA9Def4sec Arman 4 Box1.png|Cryo-EM image of an intact ARMAN cell from an Iron Mountain biofilm. Image width is 576 nm. File:CroV TEM (cropped).jpg|Cryo-EM image of the CroV giant marine virus(scale bar represents 200 nm) == See also ==