Cryoelectron+Microscopy

Cryoelectron Microscopy

Cryo-Electron Microscopy is a cell biological technique that allows to see the specimen in its natural state. This technique has been beneficial in the structural biology because when coming addition cryo techniques a 3D image of the molecule can be taken. The technique behind this is that a thin aqueous solution on a microscope grid. Then it is frozen extremely quickly to form vitreous ice. The organelle or molecule can now be viewed with out any staining, drying, or fixation. (Biology Cell) The three techniques needed in order to form the 3D image are single particle electron microscopy (cryo - EM), cryo-electron tomography (cryo - ET), and cryo - electron crystallography (cryo - EC). Cryo - ET is actually the only 3D images of cells and their organelles in their native state at a molecular resolution. (Masters, 2009) The cryotechnique was first stubled upon by Dubochet and McDowall in 1981. They were actually trying to find a better way to preserve a specimen. This can be done by aqueous solutions that are frozen in a vitreous state or using thin layers of water. (Dubochet, et. al., 1982) Related Research

In the first article “Flexible Fitting of High-Resolution X-Ray Structures into Cryoelectron Microscopy Maps Using Biased Molecular Dynamics Simulations,” Orchezowski and Tama are trying to further advance the cryoelectron technique. In the article they are trying to get a high resolution structure of a molecule by combining the cryo electron density map with a all atom high resoution to the same structure. Using these new methods Orchezowski and Tama were able to arrive at the higher resolution in cryo-EM. (Orchezowski and Tama, 2008)

The second article, “Cryoelectron microscopy and cryoelectron tomography of the nuclear pre-mRNA processing machine” they are studying the precursor messenger RNA (pre - mRNA) by using the cryo EM technique. Analyzing pre -mRNA in a frozen - hydrated state is beneficial for studying it due to the fact that it will be in its native state. Also when combing this technique with cryoelectron tomography we can see the object in 3D, which proves to be beneficial in this article. When looking at the ice-embedded large nuclear ribonucleoprotein (lnRNP) in a negative stain they could see holes, but when the image is in 3D they could actually see little fibers connecting these holes. In addition to that they were able to find a fifth structure on the lnRNP when previous studies have suggested that there was only four. Using these state of the art techniques can be advantageous in new studies. (Medalia et. al., 2002)

The third article that uses the cryoelectron microscopy technique is "Identification of the β 1-integrin binding site on α -actinin by cryoelectron microscopy.” Kelly and Taylor are binding a peptide to a lipid monolayer and have success using this technique. They come to the conclusion that the the β 1-integrin cytoplasmic domain binds α -actinin between the first and second, 3-helix motifs in the central rod domain. (Kelly and Taylor, 2005)

Dubochet, J, J Lepault, R Freeman, J A. Berriman, and J C. Homo. "Electron microscopy of frozen water and aqueous solutions." //Journal of Microscopy// 128.3 Dec. (1982): 219-37.

Kelly, D F., and K a. Taylor. "Identification of the β 1-integrin binding site on α -actinin by cryoelectron microscopy." //Journal of Structural Biology// 139.3 Mar. (2005): 290- 302.

Masters, Barry R (March 2009) History of the ElectronMicroscope in Cell Biology. In: Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester.

Medalia, O, D Typke, R Hegerl, M Angenitzki, and J Sperling. "Cryoelectron microscopy and cryoelectron tomography of the nuclear pre-mRNA processing machine." //Journal of Structural Biology// 138.1-2 May (2002): 78-84.

Orzechowski, M, and F Tama. "Flexible Fitting of High-Resolution X-Ray Structures into Cryoelectron Microscopy Maps Using Biased Molecular Dynamics Simulations." //Biophysical Journal// 95.1215 Dec. (2008): 5692-5705