Cryo-Electron Microscopy: A Powerful Tool for Visualizing the Structure of Macromolecules
Cryo-Electron Microscopy: A Powerful Tool for Visualizing the Structure of Macromolecules
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Cryo-electron microscopy (cryo-EM) is a technique used to obtain high-resolution structural information about biomolecules. While electron microscopy has been used since the 1930s to image thin biological specimens, the development of cryo-EM in the late 20th century revolutionized structural biology. By rapidly freezing biomolecules embedded in a thin layer of vitreous ice, cryo-EM allows imaging of specimens close to their native hydrated state. This preserves their three-dimensional structure at near-atomic resolution.
Cryo Electron Microscopy works on the principle that a beam of electrons can be focused into a thin stream using electromagnetic lenses, just like light microscopes. But at much higher resolution due to the smaller de Broglie wavelength of electrons. The specimen is bombarded with electrons and an image is formed based on how the electrons interact with the sample; transmitted electrons form the image. Computer processing is then used to combine images of identical molecules oriented in different positions, enhancing fine structural details.
Sample Preparation for Cryo-EM
Proper sample preparation is crucial for obtaining high-quality cryo-EM data. Purified macromolecules are typically suspended in buffer at micromolar concentrations. A thin film of this solution is then applied to a copper grid coated with a perforated carbon support film. Before the water can evaporate and destroy the molecular structure, the grid is plunged into liquid ethane, cooled to near liquid nitrogen temperatures, at a controlled rate.
This flash-freezes the sample, trapping the molecules embedded in a thin layer of amorphous ice without forming ice crystals. The ice layer must be thin enough for electrons to transmit through but thick enough to protect the molecules from radiation damage during imaging. Adjusting sample concentration, temperature, and humidity optimizes freezing conditions for different specimens. The frozen hydrated grids can then be stored in liquid nitrogen until loaded into the cryo-EM.
Components of a Cryo-EM
A basic cryo-EM consists of several components to facilitate sample imaging. The electron source, typically a field emission gun, emits electrons which are focused by a system of electromagnetic lenses into a beam. Specimen holders maintain grids in the vacuum chamber at liquid nitrogen temperatures to prevent sample degradation. Computer-controllable stage systems allow automated navigation and high-precision imaging of targeted areas on the grid.
Powerful microscopes magnify the resultant image, which is detected on a direct electron detector camera. For single-particle analysis, cryo-EMs are often equipped with a voltage variable phase plates that enhance contrast of projections from loosely specified particle orientations. Other accessories like electron energy loss spectrometers enable elemental mapping and energy-filtered imaging. Integration of extensive computing hardware supports the complex image processing required for high-resolution 3D structure determination.
Image Formation and Single-Particle Reconstruction
When the electron beam interacts with a sample, some electrons are scattered while others pass through undeflected. The unscattered electrons form the image, appearing brighter where the sample is thin. Projection images of identical molecules in random orientations are automatically collected. Using reference-free algorithms, individual particle images are selected, aligned based on common structural features, and classified into representative groups.
The orientations of each particle are then determined relative to a common coordinate system. The oriented data is used to computationally reconstruct the three-dimensional structure in an iterative process. Constraints are applied so that projections of the model match the experimental images as closely as possible. With a large enough dataset, macromolecular complexes can typically be resolved to better than 4 angstroms, revealing atomic details like the arrangement of amino acids in a protein.
Expanding Applications of Cryo-EM
Once limited to relatively large particles over 200 kDa, steady technical advances now allow cryo-EM structure determination of smaller targets down to single proteins. Coupled with automation and artificial intelligence for image analysis, cryo-EM is being applied to many new biological questions. Examples include visualizing transient protein states, mapping conformational changes, locating bound ligands, and analyzing heterogeneous complexes. Virus structure elucidation has provided insight into infection pathways.
Moreover, the technique is well-suited to studying challenging, non-crystalline samples. Near-atomic cryo-EM structures of membrane proteins in their native lipid bilayer environment reveal mechanisms of transport and signaling. Intact cells or tissues may even be imaged once resolutions improve. Cryo-EM sits at the forefront of structural biology, qualitatively expanding our visual understanding of life at the molecular level. Widespread availability and training are democratizing this transformative technology across many disciplines.
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