Transmission electron cryomicroscopy

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CryoTEM image of GroEL suspended in amorphous ice at 50000x magnification Cryoem groel.jpg
CryoTEM image of GroEL suspended in amorphous ice at 50000× magnification
Structure of Alcohol oxidase from Pichia pastoris by CryoTEM

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 (generally liquid-nitrogen temperatures). [1] Cryo-EM, specifically 3-dimensional electron microscopy (3DEM), is gaining popularity in structural biology. [2]

Contents

The utility of transmission electron cryomicroscopy stems from the fact that it allows the observation of specimens that have not been stained or fixed in any way, showing them in their native environment. This is in contrast to X-ray crystallography, which requires crystallizing the specimen, which can be difficult, and placing them in non-physiological environments, which can occasionally lead to functionally irrelevant conformational changes.

Advances in electron detector technology, particularly DED (Direct Electron Detectors) as well as more powerful software imaging algorithms have allowed for the determination of macromolecular structures at near-atomic resolution. [3] Imaged macromolecules include viruses, ribosomes, mitochondria, ion channels, and enzyme complexes. Starting in 2018, cryo-EM could applied to structures as small as hemoglobin (64 kDa) [4] and with resolutions up to 1.8 Å. [5] In 2019, cryo-EM structures represented 2.5% of structures deposited in the Protein Data Bank, [6] and this number continues to grow. [7] An application of cryo-EM is cryo-electron tomography (cryo-ET), where a 3D reconstruction of the sample is created from tilted 2D images.

Development

The original rationale for CryoTEM was as a means to fight radiation damage for biological specimens. The amount of radiation required to collect an image of a specimen in the electron microscope is high enough to be a potential source of specimen damage for delicate structures. In addition, the high vacuum required on the column of an electron microscope makes the environment for the sample quite harsh.

The problem of the vacuum was partially solved by the introduction of negative stains but even with negative stains biological samples are prone to structural collapse upon dehydration of the specimen. Embedding the samples in ice below the sublimation temperature was a possibility that was contemplated early on, but water tends to arrange into a crystalline lattice of lower density upon freezing and this can destroy the structure of anything that is embedded in it.

In the early 1980s, several groups studying solid state physics were attempting to produce vitreous ice by different means, such as high pressure freezing or flash freezing. In a seminal paper in 1984, the group led by Jacques Dubochet at the European Molecular Biology Laboratory showed images of adenovirus embedded in a vitrified layer of water. [8] 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 (80–300 kV) is high enough that covalent bonds can be broken. When imaging specimens are vulnerable to radiation damage, 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. [1] [2]

In 2015, Bridget Carragher and colleagues at the Scripps National Resource for Automated Molecular Microscopy used techniques she and Clint Potter developed to determine the first cryo-EM structure with a resolution finer than 3 Å, thereby elevating CryoTEM as a tool comparable to and potentially superior to traditional x-ray crystallography techniques. [9] [10] Since then, higher resolutions have been achieved, including a 2.2 Å structure of bacterial enzyme β-galactosidase in 2015 [11] and a 1.8 Å structure of glutamate dehydrogenase in 2016. [12] Cryo-EM has also been used to determine the structure of various viruses, including the Zika virus, [13] and has been applied to large complexes such as the spliceosome. [14] In 2017, the Nobel Prize in Chemistry was awarded jointly to Jacques Dubochet, Joachim Frank and Richard Henderson, "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution". [15]

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, [16] and sulfur, which is prone to sublimation in the vacuum of electron microscopes, can be stabilized and imaged in CryoTEM. [17]

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. [18] There are two popular approaches that are widely used nowadays in cryo-EM image processing, the maximum likelihood approach that was discovered in 1998 [19] and relatively recently adapted Bayesian approach. [20]

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. [21] 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. [22]

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. [21] The smoothing of the maps occurs through assuming prior probability to be a Gaussian distribution and analyzing the data in the Fourier space. Since the connection between the prior knowledge and the dataset is established, there is less chance for human factor errors which potentially increases the objectivity of image reconstruction. [20]

With emerging new methods of cryo-TEM imaging and image reconstruction the new software solutions appear that help to automate the process. After the empirical Bayesian approach have been implemented in the open source computer program RELION (REgularized LIkelihood OptimizatioN) for 3D reconstruction, [23] [24] 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. [25] [26]

Techniques

A variety of techniques can be used in CryoTEM. [27] Popular techniques include:

  1. Electron crystallography
    1. Analysis of two-dimensional crystals
    2. Analysis of helical filaments or tubes
    3. Microcrystal Electron Diffraction (MicroED) [28] [29] [30] [31]
  2. Single particle analysis (SPA)
    1. Time-resolved CryoTEM [32] [33] [34]
  3. Electron cryotomography (cryoET)

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 is a field that is many centuries old which, as defined by the Journal of Structural Biology, deals with structural analysis of living material at every level of organization. Early structural biologists throughout the 19th and early 20th centuries were primarily only able to study structures to the limit of the naked eye's visual acuity and through magnifying glasses and light microscopes.

Electron crystallography is a method to determine the arrangement 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">Electron cryotomography</span>

Cryo-electron tomography (cryo-ET) is an imaging technique used to produce high-resolution (~1–4 nm) three-dimensional views of samples, often biological macromolecules and cells. cryo-ET 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.

The EM Data Bank or Electron Microscopy Data Bank (EMDB) collects 3D EM maps and associated experimental data determined using electron microscopy of biological specimens. It was established in 2002 at the MSD/PDBe group of the European Bioinformatics Institute (EBI), where the European site of the EMDataBank.org consortium is located. As of 2015, the resource contained over 2,600 entries with a mean resolution of 15Å.

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

Electron tomography (ET) is a tomography technique for obtaining detailed 3D structures of sub-cellular, macro-molecular, or materials specimens. Electron tomography is an extension of traditional transmission electron microscopy and uses a transmission electron microscope to collect the data. In the process, a beam of electrons is passed through the sample at incremental degrees of rotation around the center of the target sample. This information is collected and used to assemble a three-dimensional image of the target. For biological applications, the typical resolution of ET systems are in the 5–20 nm range, suitable for examining supra-molecular multi-protein structures, although not the secondary and tertiary structure of an individual protein or polypeptide. Recently, atomic resolution in 3D electron tomography reconstructions has been demonstrated.

Resolution in terms of electron density is a measure of the resolvability in the electron density map of a molecule. In X-ray crystallography, resolution is the highest resolvable peak in the diffraction pattern, while resolution in cryo-electron microscopy is a frequency space comparison of two halves of the data, which strives to correlate with the X-ray definition.

Cryofixation is a technique for fixation or stabilisation of biological materials as the first step in specimen preparation for electron microscopy and cryo-electron microscopy. Typical specimens for cryofixation include small samples of plant or animal tissue, cell suspensions of microorganisms or cultured cells, suspensions of viruses or virus capsids and samples of purified macromolecules, especially proteins.

In structural biology, as well as in virtually all sciences that produce three-dimensional data, the Fourier shell correlation (FSC) measures the normalised cross-correlation coefficient between two 3-dimensional volumes over corresponding shells in Fourier space (i.e., as a function of spatial frequency). The FSC is the three-dimensional extension of the two-dimensional Fourier ring correlation (FRC); also known as: spatial frequency correlation function.

<span class="mw-page-title-main">Single particle analysis</span>

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 induced coupled plasma mass spectroscopy (ICP-MS).

<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">Cryogenic electron microscopy</span> Form of transmission electron microscopy (TEM)

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. 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 for macromolecular structure determination without the need for crystallization.

<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.

<span class="mw-page-title-main">Sjors Scheres</span>

Sjors Hendrik Willem ScheresFRS is a Dutch scientist at the MRC Laboratory of Molecular Biology Cambridge, UK.

<span class="mw-page-title-main">Bridget Carragher</span> American physicist

Bridget Olivia Carragher is a South African physicist specialized in electron microscopy.

Microcrystal electron diffraction, or MicroED, is a CryoEM method that was developed by the Gonen laboratory in late 2013 at the Janelia Research Campus of the Howard Hughes Medical Institute. MicroED is a form of electron crystallography where thin 3D crystals are used for structure determination by electron diffraction. Prior to this demonstration, macromolecular (protein) electron crystallography was only used on 2D crystals, for example.

<span class="mw-page-title-main">Kiyoshi Nagai</span> Japanese structural biologist (1949–2019)

Kiyoshi Nagai was a Japanese structural biologist at the MRC Laboratory of Molecular Biology Cambridge, UK. He was known for his work on the mechanism of RNA splicing and structures of the spliceosome.

Mavis Agbandje-McKenna was a Nigerian-born British medical biophysicist, structural virologist, and a professor of structural biology, as well as the director of the Center for Structural Biology at the University of Florida in Gainesville, Florida. Agbandje-McKenna studied parvovirus structures using X-ray crystallography and cryogenic electron microscopy and did much of the initial work to elucidate the basic structure and function of adeno-associated viruses (AAVs). Her viral characterization and elucidation of antibody binding sites on AAV capsids has led to the development of viral capsid development and gene therapy approaches that evade immune detection and can be used to treat human diseases such as muscular dystrophies. Agbandje-McKenna was recognized with the 2020 American Society of Gene and Cell Therapy Outstanding Achievement Award for her contributions to the field. She died in 2021 from amyotrophic lateral sclerosis.

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