Transmission electron cryomicroscopy

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CryoTEM image of GroEL suspended in amorphous ice at 50000× magnification
Structure of Alcohol oxidase from Pichia pastoris by CryoTEM

Transmission electron cryomicroscopy (CryoTEM) (also 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] CryoTEM is gaining popularity in structural biology.[2]

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.

The resolution of CryoTEM maps is improving steadily, and in 2014 some structures at near-atomic resolution had been obtained, including those of viruses, ribosomes, mitochondria, ion channels, and enzyme complexes as small as 170 kDa at a resolution of 4.5 Å.[1] Bridget Carragher and colleagues at the Scripps National Resource for Automated Molecular Microscopy used techniques she and Clint Potter developed to create the first transmission electron cryomicroscopy structural biology image with a resolution finer than 3 Ångströms, thereby elevating CryoTEM as a tool comparable to and potentially superior to traditional x-ray crystallography techniques.[3][4] A 2.2 Å map of a bacterial enzyme beta-galactosidase was published in June 2015.[5] An application of CryoTEM is electron cryotomography (CryoET), where a 3D reconstruction of a sample is created from tilted 2D images.


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 '80s, 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.[6] This paper is generally considered to mark the origin of CryoTEM, 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 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 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".[7]

Biological specimens[edit]

Thin film[edit]

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[edit]

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[edit]

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,[8] and sulfur, which is prone to sublimation in the vacuum of electron microscopes, can be stabilized and imaged in CryoTEM.[9]


A variety of techniques can be used in CryoTEM.[10] Popular techniques include:

  1. Electron crystallography
    1. Analysis of two-dimensional crystals
    2. Analysis of helical filaments or tubes
  2. Single particle analysis
  3. Electron cryotomography
  4. MicroED[11][12][13][14]
  5. Time-resolved CryoTEM[15][16][17]

See also[edit]


  1. ^ a b c Kuehlbrandt, Werner (2014). "Cryo-EM enters a new era". eLife. 3: e03678. doi:10.7554/elife.03678. PMC 4131193. PMID 25122623.
  2. ^ a b Callaway, Ewen (2015). "The revolution will not be crystallized: A new method sweeps through structural biology". Nature. 525 (7568): 172–4. Bibcode:2015Natur.525..172C. doi:10.1038/525172a. PMID 26354465.
  3. ^ Dellisanti, Cosma (2015). "A barrier-breaking resolution". Nature Structural & Molecular Biology. 22 (5): 361. doi:10.1038/nsmb.3025.
  4. ^ Campbell, Melody G; Veesler, David; Cheng, Anchi; Potter, Clinton S; Carragher, Bridget (2015). "2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy". eLife. 4. doi:10.7554/eLife.06380. PMC 4391500. PMID 25760083.
  5. ^ Bartesaghi, A.; Merk, A.; Banerjee, S.; Matthies, D.; Wu, X.; Milne, J. L. S.; Subramaniam, S. (2015). "2.2 a resolution cryo-EM structure of -galactosidase in complex with a cell-permeant inhibitor". Science. 348 (6239): 1147–51. Bibcode:2015Sci...348.1147B. doi:10.1126/science.aab1576. PMID 25953817.
  6. ^ Adrian, Marc; Dubochet, Jacques; Lepault, Jean; McDowall, Alasdair W. (1984). "Cryo-electron microscopy of viruses". Nature. 308 (5954): 32–6. Bibcode:1984Natur.308...32A. doi:10.1038/308032a0. PMID 6322001.
  7. ^ "The 2017 Nobel Prize in Chemistry - Press Release". 4 October 2017. Retrieved 4 October 2017.
  8. ^ Zachman, Michael J.; Asenath-Smith, Emily; Estroff, Lara A.; Kourkoutis, Lena F. (2016). "Site-Specific Preparation of Intact Solid–Liquid Interfaces by Label-Free in Situ Localization and Cryo-Focused Ion Beam Lift-Out". Microscopy and Microanalysis. 22 (6): 1338–1349. Bibcode:2016MiMic..22.1338Z. doi:10.1017/S1431927616011892. PMID 27869059.
  9. ^ Levin, Barnaby D.A.; Zachman, Michael J.; Werner, Jörg G.; Sahore, Ritu; Nguyen, Kayla X.; Han, Yimo; Xie, Baoquan; Ma, Lin; Archer, Lynden A.; Giannelis, Emmanuel P.; Wiesner, Ulrich; Kourkoutis, Lena F.; Muller, David A. (2017). "Characterization of Sulfur and Nanostructured Sulfur Battery Cathodes in Electron Microscopy Without Sublimation Artifacts". Microscopy and Microanalysis. 23 (1): 155–162. Bibcode:2017MiMic..23..155L. doi:10.1017/S1431927617000058. PMID 28228169.
  10. ^ Presentation on Cryoelectron Microscopy |
  11. ^ Shi, Dan; Nannenga, Brent L.; Iadanza, Matthew G.; Gonen, Tamir (2013-11-19). "Three-dimensional electron crystallography of protein microcrystals". eLife. 2: e01345. doi:10.7554/eLife.01345. ISSN 2050-084X. PMC 3831942. PMID 24252878.
  12. ^ Nannenga, Brent L.; Shi, Dan; Leslie, Andrew G. W.; Gonen, Tamir (2014). "High-resolution structure determination by continuous-rotation data collection in MicroED". Nature Methods. 11 (9): 927–930. doi:10.1038/nmeth.3043. ISSN 1548-7105. PMC 4149488. PMID 25086503.
  13. ^ Shi, Dan; Nannenga, Brent L.; de la Cruz, M. Jason; Liu, Jinyang; Sawtelle, Steven; Calero, Guillermo; Reyes, Francis E.; Hattne, Johan; Gonen, Tamir (2016). "The collection of MicroED data for macromolecular crystallography". Nature Protocols. 11 (5): 895–904. doi:10.1038/nprot.2016.046. ISSN 1750-2799. PMC 5357465. PMID 27077331.
  14. ^ de la Cruz, M. Jason; Hattne, Johan; Shi, Dan; Seidler, Paul; Rodriguez, Jose; Reyes, Francis E.; Sawaya, Michael R.; Cascio, Duilio; Weiss, Simon C. (2017-02-13). "Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED". Nature Methods. 14 (4): 399–402. doi:10.1038/nmeth.4178. ISSN 1548-7105. PMC 5376236. PMID 28192420.
  15. ^ Fu, Ziao; Kaledhonkar, Sandip; Borg, Anneli; Sun, Ming; Chen, Bo; Grassucci, Robert A.; Ehrenberg, Måns; Frank, Joachim (2016). "Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy". Structure. 24 (12): 2092–2101. doi:10.1016/j.str.2016.09.014. PMC 5143168. PMID 27818103.
  16. ^ Feng, Xiangsong; Fu, Ziao; Kaledhonkar, Sandip; Jia, Yuan; Shah, Binita; Jin, Amy; Liu, Zheng; Sun, Ming; Chen, Bo; Grassucci, Robert A.; Ren, Yukun; Jiang, Hongyuan; Frank, Joachim; Lin, Qiao (2017). "A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM". Structure. 25 (4): 663–670.e3. doi:10.1016/j.str.2017.02.005. PMC 5382802. PMID 28286002.
  17. ^ Chen, Bo; Kaledhonkar, Sandip; Sun, Ming; Shen, Bingxin; Lu, Zonghuan; Barnard, David; Lu, Toh-Ming; Gonzalez, Ruben L.; Frank, Joachim (2015). "Structural Dynamics of Ribosome Subunit Association Studied by Mixing-Spraying Time-Resolved Cryogenic Electron Microscopy". Structure. 23 (6): 1097–105. doi:10.1016/j.str.2015.04.007. PMC 4456197. PMID 26004440.

Further reading[edit]

  • Frank, Joachim (2006). Three-Dimensional Electron Microscopy of Macromolecular Assemblies. New York: Oxford University Press. ISBN 0-19-518218-9.
  • Van Heel, Marin; Gowen, Brent; Matadeen, Rishi; Orlova, Elena V.; Finn, Robert; Pape, Tillmann; Cohen, Dana; Stark, Holger; Schmidt, Ralf; Schatz, Michael; Patwardhan, Ardan (2000). "Single-particle electron cryo-microscopy: Towards atomic resolution". Quarterly Reviews of Biophysics. 33 (4): 307–69. doi:10.1017/s0033583500003644. PMID 11233408.

External links[edit]