Cryoelectron microscopy

Electron diffraction studies are usually limited to transferred films (see Chapter XV), One study on Langmuir films of fatty acids has used cryoelectron microscopy to fix the structures on vitrified water [179], Electron diffraction from these layers showed highly twinned structures in the form of faceted crystals. 

PDB files were designed for storage of crystal structures and related experimental information on biological macromolecules, primarily proteins, nucleic acids, and their complexes. Over the years the PDB file format was extended to handle results from other experimental (NM.R, cryoelectron microscopy) and theoretical methods... 

Alphaviruses, such as Sindbis virus and Semliki Forest virus, are a group of mosquito-borne, enveloped RNA viruses that can cause encephalitis, fever, arthritis and rashes in mammals. These viruses have two protein shells—an outer glycoprotein layer and an inner core— which are separated by a lipid bilayer, a membrane. Studies by cryoelectron microscopy have shown that... 

The conformation change in the power stroke has been studied in two ways (1) cryoelectron microscopy together with computerized image analysis... 

Figure 3.3 Molecular structure of G-protein-coupled receptors. In (a) the electron density map of bovine rhodopsin is shown as obtained by cryoelectron microscopy of two-dimensional arrays of receptors embedded in lipid membrane. The electron densities show seven peaks reflecting the seven a-helices which are predicted to cross the cell membrane. In (b) is shown a helical-wheel diagram of the receptor orientated according to the electron density map shown in (a). The diagram is seen as the receptor would be viewed from outside the cell membrane. The agonist binding pocket is illustrated by the hatched region between TM3, TM5 and TM6. (From Schertler et al. 1993 and Baldwin 1993, reproduced from Schwartz 1996). Reprinted with permission from Textbook of Receptor Pharmacology. Eds Foreman, JC and Johansen, T. Copyright CRC Press, Boca Raton, Florida...

M.Geinmi Precession electron diffraction in EUROSUMMER SCHOOL Electron Crystalloggraphy and cryoelectron microscopy on inorganic materials and organic and biological molecules , Barcelona (Spain), July 23-27,2001... 

Furrer, P., Bednar, I., Dubochet, I., Hamiche, A., and Prunell, A. (1995) DNA at the entry-exit of the nucleosome observed by cryoelectron microscopy. I. Struct. Biol. 114, 177-183. 

Bednar, J., Horowitz, R.A., Dubochet, J., and Woodcock, C.L. (1995) Chromatin conformation and salt-induced compaction three-dimensional structural information from cryoelectron microscopy. J. Cell Biol. 131, 1365-1376. 

Cryoelectron microscopy makes it possible to have a direct view into the frozen sample without additional preparation [100]. With the aid of a cryogen (e.g., liquid nitrogen-cooled liquid ethane), the sample is plunge frozen as a very thin aqueous film prepared on a microscopic grid. Subsequently, the vitrified specimen is directly transferred into a precooled electron microscope. Because the specimens are usually ) 2005 by CRC Press LLC... 

Harris J.R., Negative Staining and Cryoelectron Microscopy The Thin Film Techniques, Bios Scientific, Oxford, 1997. 

A number of factors for DOTAP-cholesterol/DNA complex preparation including the DNA/liposome ratio, mild sonication, heating, and extrusion were found to be crucial for improved systemic delivery maximal gene expression was obtained when a homogeneous population of DNA/liposome complexes (200-450 nm) was used. Cryoelectron microscopy showed that the DNA was condensed on the interior of liposomes between two lipid bilayers in these formulations, a factor that was thought to be responsible for the high transfection efficiency in vivo and for the broad tissue distribution (150). 

Another striking paper in which image reconstruction with cryoelectron microscopy yields a model of the PDH complex. Compare this model with that in the paper by Milne et al. 

McGough, A., Way, M., and DeRosier, D. (1994). Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis. 

Tang, J., Taylor, D. W., and Taylor, K. A. (2001). The three-dimensional structure of alpha-actinin obtained by cryoelectron microscopy suggests a model for Ca(2+)-dependent actin binding. / Mol. Biol. 310, 845-858. 

Bottcher, C., Endisch, C., Fuhrhop, J.-H., Catterall, C. and Eaton, M. (1998) High-yield preparation of oligomeric C-type DNA toroids and their characterization by cryoelectron microscopy J. Am. Chem. Soc., 120, 12-17. 

A detailed view of the kinesin-microtubule complex has been obtained by combining high-resolution structures of the individual components from X-ray crystallography (kinesin) and electron diffraction (tubulin Lowe et al., 2001 Nogales et al, 1998) with low-resolution models of kinesin-decorated microtubules obtained by cryoelectron microscopy and image reconstruction (Hirose et al, 1999 Hoenger et al., 2000 Kikkawa et al, 2001 Kozielski et al., 1998 Rice et al., 1999 Skiniotis et al., 2003 Wendt... 

In cryoelectron microscopy, the micelles can be examined in the frozen hydrated state without staining. Van Bruggen etal. (1986) in a preliminary report, showed micrographs of particles with a type of substructure similar to that seen by other methods (Fig. 12d). 

The first structural location of the taxane binding site [42] placed it in the interprotofilament space, thus supporting the biochemical results. However, this changed when the first high resolution 3D structure of the paclitaxel-tubulin complex was solved by electron-crystallography of a two-dimensional zinc-induced tubulin polymer [5]. The fitting of this structure into a three-dimensional reconstruction of microtubules from cryoelectron microscopy allowed a pseudo atomic resolution model of microtubules [43] in which the paclitaxel binding site was placed inside the lumen of the microtubules hidden from the outer solvent. 

In addition, docking the EC-derived near-atomic resolution structures of tubulin into cryoelectron microscopy maps of MT, has provided more precise insights into the structure of MT, their polymerization dynamics and the way how normal MT behavior is disrupted by antimitotic drugs [23, 24],... 

The first near-atomic resolution structure of the tubulin dimer was determined from a 3.7-A electron density map obtained by EC from the two-dimensional, crystalline sheets of tubulin that form in the presence of Zn ions and PTX [11], This polymerized form of tubulin consists of protofilaments arranged in an antiparallel way, while protofilaments are parallel in genuine MT. Despite this different orientation of adjacent protofilaments, 3D reconstruction of intact MT from cryoelectron microscopy images at 20 A or 8 A resolution demonstrate that the conformation of tubulin is... 

The two structures of the tubulin/EpoA complex derived from EC and NMR data are significantly different, although both are able to explain many available SAR data. It is worth noting that both structures were derived from samples that were not microtubules. The EC data were obtained from 2D sheets of antiparallel protofilaments, while MT are formed of parallel protofilaments arranged into cylinders. In fact, the position of the M loop differs in the two tubulin forms, as shown by docking of the EC-derived structure of PTX/MT into the 8 A cryoelectron microscopy images of MT [24],... 

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