Cryogenic electron microscopy

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Titan Krios at the University of Leeds Titan Krios University of Leeds.jpg
Titan Krios at the University of Leeds

Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on 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. [1] 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. [2] This has attracted wide attention to the approach as an alternative to X-ray crystallography or NMR spectroscopy for macromolecular structure determination without the need for crystallization. [3]

Contents

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." [4] Nature Methods also named cryo-EM as the "Method of the Year" in 2015. [5]

History

Early development

In the 1960s, the use of transmission electron microscopy for structure determination methods 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. [6] Both liquid helium (−269  °C or 4  K or −452.2  °F) and liquid nitrogen (−195.79 °C or 77 K or −320 °F) were considered as cryogens. 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. [7]

However, these results were not reproducible and amendments were published in Nature just two years later informing 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", [8] than what was previously stated.

In 1981, Alasdair McDowall and Jacques Dubochet, scientists at the European Molecular Biology Laboratory, reported the first successful implementation of cryo-EM. [9] McDowall and Dubochet vitrified pure water in a thin film by spraying it 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 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. [10]

Recent advancements

The 2010s were marked with drastic advancements of electron cameras. Notably, the improvements made to direct electron detectors have led to a "resolution revolution" [11] pushing the resolution barrier beneath the crucial ~2-3 Å limit to resolve amino acid position and orientation. [12]

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). [11]

More recently, advancements in the use of protein-based imaging scaffolds are helping to solve the problems of sample orientation bias and size limit. Proteins smaller than ~50 kDa generally have insufficient SNR to be able to resolve protein particles in the image, making 3D reconstruction difficult or impossible. [13] Imaging scaffolds boost the SNR of smaller proteins by binding them to a larger object, the scaffold. The Yeates group at UCLA were able to create a clearer image of three variants of KRAS (roughly 19 kDa in size) by utilising a rigidified imaging scaffold, and using DARPins as modular binding domain between the scaffold and the protein-of-interest. [14]

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." [4]

Comparisons to X-ray crystallography

Traditionally, X-ray crystallography has been the most popular technique for determining the 3D structures of biological molecules. [15] 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. [16] 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. [16]

The resolution of X-ray crystallography is limited by crystal homogeneity, [17] 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. [18] 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, [19] [20] 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. [21]

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, [17] and the highest resolution achieved on record (as of September 30, 2022) is 0.48 Å. [22] As of 2020, the majority of the protein structures determined by cryo-EM are at a lower resolution of 3–4 Å. [23] However, as of 2020, the best cryo-EM resolution has been recorded at 1.22 Å, [20] making it a competitor in resolution in some cases.

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. [24]

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.

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.

Centers

The Federal Institute of Technology, the University of Lausanne and the University of Geneva opened the Dubochet Center For Imaging (DCI) at the end of November 2021, for the purposes of applying and further developing cryo-EM. [25] Less than a month after the first identification of the SARS-CoV-2 Omicron variant, researchers at the DCI were able to define its structure, identify the crucial mutations to circumvent individual vaccines and provide insights for new therapeutic approaches. [26]

The Danish National cryo-EM Facility also known as EMBION was inaugurated on December 1, 2016. EMBION is a cryo-EM consortium between Danish Universities (Aarhus University host and University of Copenhagen co-host).

Single particle analysis workflow Cryogenic electron microscopy workflow.svg
Single particle analysis workflow

Advanced methods

See also

Related Research Articles

<span class="mw-page-title-main">Electron microscope</span> Type of microscope with electrons as a source of illumination

An electron microscope is a microscope that uses a beam of electrons as a source of illumination. They use electron optics that are analogous to the glass lenses of an optical light microscope to control the electron beam, for instance focusing them to produce magnified images or electron diffraction patterns. As the wavelength of an electron can be up to 100,000 times smaller than that of visible light, electron microscopes have a much higher resolution of about 0.1 nm, which compares to about 200 nm for light microscopes. Electron microscope may refer to:

<span class="mw-page-title-main">Structural biology</span> Study of molecular structures in biology

Structural biology, as defined by the Journal of Structural Biology, deals with structural analysis of living material at every level of organization.

Electron crystallography is a subset of methods in electron diffraction focusing just upon detailed determination of the positions of atoms in solids using a transmission electron microscope (TEM). It can involve the use of high-resolution transmission electron microscopy images, electron diffraction patterns including convergent-beam electron diffraction or combinations of these. It has been successful in determining some bulk structures, and also surface structures. Two related methods are low-energy electron diffraction which has solved the structure of many surfaces, and reflection high-energy electron diffraction which is used to monitor surfaces often during growth.

<span class="mw-page-title-main">Transmission electron cryomicroscopy</span>

Transmission electron cryomicroscopy (CryoTEM), commonly known as cryo-EM, is a form of cryogenic electron microscopy, more specifically a type of transmission electron microscopy (TEM) where the sample is studied at cryogenic temperatures. Cryo-EM, specifically 3-dimensional electron microscopy (3DEM), is gaining popularity in structural biology.

<span class="mw-page-title-main">Cryogenic electron tomography</span>

Cryogenic electron tomography (cryoET) is an imaging technique used to reconstruct high-resolution (~1–4 nm) three-dimensional volumes of samples, often biological macromolecules and cells. cryoET is a specialized application of transmission electron cryomicroscopy (CryoTEM) in which samples are imaged as they are tilted, resulting in a series of 2D images that can be combined to produce a 3D reconstruction, similar to a CT scan of the human body. In contrast to other electron tomography techniques, samples are imaged under cryogenic conditions. For cellular material, the structure is immobilized in non-crystalline, vitreous ice, allowing them to be imaged without dehydration or chemical fixation, which would otherwise disrupt or distort biological structures.

<span class="mw-page-title-main">Richard Henderson (biologist)</span> British biologist

Richard Henderson is a British molecular biologist and biophysicist and pioneer in the field of electron microscopy of biological molecules. Henderson shared the Nobel Prize in Chemistry in 2017 with Jacques Dubochet and Joachim Frank. "Thanks to his work, we can look at individual atoms of living nature, thanks to cryo-electron microscopes we can see details without destroying samples, and for this he won the Nobel Prize in Chemistry."

<span class="mw-page-title-main">Eva Nogales</span> Biophysicist, professor

Eva Nogales is a Spanish-American biophysicist at the Lawrence Berkeley National Laboratory and a professor at the University of California, Berkeley, where she served as head of the Division of Biochemistry, Biophysics and Structural Biology of the Department of Molecular and Cell Biology (2015–2020). She is a Howard Hughes Medical Institute investigator.

Resolution in the context of structural biology is the ability to distinguish the presence or absence of atoms or groups of atoms in a biomolecular structure. Usually, the structure originates from methods such as X-ray crystallography, electron crystallography, or cryo-electron microscopy. The resolution is measured of the "map" of the structure produced from experiment, where an atomic model would then be fit into. Due to their different natures and interactions with matter, in X-ray methods the map produced is of the electron density of the system, whereas in electron methods the map is of the electrostatic potential of the system. In both cases, atomic positions are assumed similarly.

Experimental approaches of determining the structure of nucleic acids, such as RNA and DNA, can be largely classified into biophysical and biochemical methods. Biophysical methods use the fundamental physical properties of molecules for structure determination, including X-ray crystallography, NMR and cryo-EM. Biochemical methods exploit the chemical properties of nucleic acids using specific reagents and conditions to assay the structure of nucleic acids. Such methods may involve chemical probing with specific reagents, or rely on native or analogue chemistry. Different experimental approaches have unique merits and are suitable for different experimental purposes.

<span class="mw-page-title-main">Single particle analysis</span> Method of analyzing transmission electron microscopy imagery

Single particle analysis is a group of related computerized image processing techniques used to analyze images from transmission electron microscopy (TEM). These methods were developed to improve and extend the information obtainable from TEM images of particulate samples, typically proteins or other large biological entities such as viruses. Individual images of stained or unstained particles are very noisy, and so hard to interpret. Combining several digitized images of similar particles together gives an image with stronger and more easily interpretable features. An extension of this technique uses single particle methods to build up a three-dimensional reconstruction of the particle. Using cryo-electron microscopy it has become possible to generate reconstructions with sub-nanometer resolution and near-atomic resolution first in the case of highly symmetric viruses, and now in smaller, asymmetric proteins as well. Single particle analysis can also be performed by inductively coupled plasma mass spectrometry (ICP-MS).

<span class="mw-page-title-main">Henning Stahlberg</span> German physicist

Henning Stahlberg is a German physicist and Professor at the Swiss Federal Institute of Technology Lausanne and the University of Lausanne, Switzerland.

<span class="mw-page-title-main">Structure validation</span> Process of evaluating 3-dimensional atomic models of biomacromolecules

Macromolecular structure validation is the process of evaluating reliability for 3-dimensional atomic models of large biological molecules such as proteins and nucleic acids. These models, which provide 3D coordinates for each atom in the molecule, come from structural biology experiments such as x-ray crystallography or nuclear magnetic resonance (NMR). The validation has three aspects: 1) checking on the validity of the thousands to millions of measurements in the experiment; 2) checking how consistent the atomic model is with those experimental data; and 3) checking consistency of the model with known physical and chemical properties.

Chikashi Toyoshima (豊島 近, Toyoshima Chikashi, born July 17, 1954) is a Japanese biophysicist. His research focuses on two proteins: the Ca2+-ATPase of muscle sarcoplasmic reticulum, and the Na+, K+-ATPase expressed in all animal cells. He is a professor at the University of Tokyo and the Foreign Associate of the National Academy of Sciences, USA. Toyoshima's research about the Ca2+-ATPase started in 1989, and within a few years of beginning this research, he and his colleagues obtained the world's first series of images of Ca2+-ATPase at the atomic level. Via x-ray crystallography, cryo-EM and other methods, he has determined the crystal structures of ten intermediates of Ca2+-ATPase. On September 10, 2015, The Royal Swedish Academy of Sciences awarded him and Poul Nissen the Gregori Aminoff Prize of 2016 for their fundamental contributions to understanding the structural basis for ATP-driven translocation of ions across membranes.

<span class="mw-page-title-main">Jacques Dubochet</span> Swiss biophysicist

Jacques Dubochet is a retired Swiss biophysicist. He is a former researcher at the European Molecular Biology Laboratory in Heidelberg, Germany, and an honorary professor of biophysics at the University of Lausanne in Switzerland.

<span class="mw-page-title-main">Tamir Gonen</span> American biochemist and biophysicist

Tamir Gonen is an American structural biochemist and membrane biophysicist best known for his contributions to structural biology of membrane proteins, membrane biochemistry and electron cryo-microscopy (cryoEM) particularly in electron crystallography of 2D crystals and for the development of 3D electron crystallography from microscopic crystals known as MicroED. Gonen is an Investigator of the Howard Hughes Medical Institute, a professor at the University of California, Los Angeles, the founding director of the MicroED Imaging Center at UCLA and a Member of the Royal Society of New Zealand.

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<span class="mw-page-title-main">Virus crystallisation</span>

Virus crystallisation is the re-arrangement of viral components into solid crystal particles. The crystals are composed of thousands of inactive forms of a particular virus arranged in the shape of a prism. The inactive nature of virus crystals provide advantages for immunologists to effectively analyze the structure and function behind viruses. Understanding of such characteristics have been enhanced thanks to the enhancement and diversity in crystallisation technologies. Virus crystals have a deep history of being widely applied in epidemiology and virology, and still to this day remains a catalyst for studying viral patterns to mitigate potential disease outbreaks.

References

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