Cryo-electron microscopy
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Electron cryomicroscopy (sometimes called cryo-EM or often cryo-electron microscopy, although it is the microscope and specimen stage and not the electrons that are cold) is a form of electron microscopy (EM) where the sample is studied at cryogenic temperatures (generally liquid nitrogen temperatures). CryoEM is developing popularity in structural biology.
A version of electron cryomicroscopy is cryo-electron tomography (CET) where a 3D reconstruction of a sample is created from tilted 2D images, again at cryogenic temperatures (either liquid nitrogen or helium).
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[edit] Biological specimens
[edit] 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 radiation sensitive, so they must be imaged with low-dose techniques (usefully, the low temperature of cryo-electron microscopy 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. This approach requires that the things being averaged are identical (e.g. ribosome particles). Analysis of ordered arrays of protein, such as 2-D crystals of membrane 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.
[edit] 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 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 cryo-electron microscopy of vitreous sections (CEMOVIS) or cryo-electron microscopy of frozen-hydrated sections.
[edit] EM structural databases
- VIPER: EM Database at Scripps
- EBI Macromolecular Structure Database at the European Bioinformatics Institute
[edit] References
- EM for Dummies. EM for Dummies. Retrieved on June 09, 2006.
- van Heel M, Gowen B, Matadeen R, Orlova EV, Finn R, Pape T, Cohen D, Stark H, Schmidt R, Schatz M, Patwardhan A (2000). "Single-particle electron cryo-microscopy: towards atomic resolution.". Q Rev Biophys. 33: 307-69.
- Frank, Joachim (2006). Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State, 2nd edition, Oxford University Press. ISBN 0195182189.
| Protein structure determination methods | ||
|---|---|---|
| High resolution: | X-ray crystallography | NMR | Electron crystallography | |
| Medium resolution: | Cryo-electron microscopy | Fiber diffraction | Mass spectrometry | |
| Spectroscopic: | NMR | Circular dichroism | Absorbance | Fluorescence | Fluorescence anisotropy | |
| Translational Diffusion: | Analytical ultracentrifugation | Size exclusion chromatography | Light scattering | NMR | |
| Rotational Diffusion: | Fluorescence anisotropy | Flow birefringence | Dielectric relaxation | NMR | |
| Chemical: | Hydrogen-deuterium exchange | Site-directed mutagenesis | Chemical modification | |
| Thermodynamic: | Equilibrium unfolding | |
| Computational: | Protein structure prediction | Molecular docking | |
| ←Tertiary structure | Quaternary structure→ | |
