Electron cryomicroscopy

A 3-D structure of an object viewed through an electron microscope is described in terms of the 3-D Coulomb potential function within the object. The image recorded in an electron microscope is a convolution of the projected potential function of the object with the contrast transfer 

According to Equation (3), when AZ is negative (overfocus) or has a small positive (underfocus) setting, 7 will have a negative value. 

The cumulative envelope function, E s), can be complex and is attributable to a number of instrumental and experimental effects, such as spatial and temporal coherence and specimen motion. It has been shown that in practice a simple Gaussian function with width B adequately describes the cumulative envelope function (Saad et al, 2001)  

Under this definition, the E function is characterized by an experimental 5 factor, which can be estimated experimentally (Saad et al, 2001). Note that the cumulative B factor applied in the final reconstruction is a composite of experimental and computational causes. The computational B factor is attributable to additional blurring effects such as inaccuracy in determining the orientation of particles, which could also be described by a Gaussian function type. The dampening of the image contrast by the 

There are several advantages of choosing higher voltages to record images. The first is the smaller chromatic aberration effect on the images and the second is the larger depth of field [see Eq. (5) below]. As pointed out above, the formulation of the currently used virus reconstruction 

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A completely new method of determining siufaces arises from the enormous developments in electron microscopy. In contrast to the above-mentioned methods where the surfaces were calculated, molecular surfaces can be determined experimentally through new technologies such as electron cryomicroscopy [188]. Here, the molecular surface is limited by the resolution of the experimental instruments. Current methods can reach resolutions down to about 10 A, which allows the visualization of protein structures and secondary structure elements [189]. The advantage of this method is that it can be apphed to derive molecular structures of maaomolecules in the native state. 

Jimenez et al. (2002) proposed a molecular model for the insulin protofilament based on these data and on electron cryomicroscopy (cryo-EM) reconstructions of insulin fibrils. The fibrils show a number of twisted morphologies that seem to be alternative packings of similar protofilaments. The protofilaments have cross sections of 30x40 A. The authors suggest a complete conversion to / -structure and model the amyloid monomer as having four jS-strands (Fig. 3B). Each insulin chain contributes two of these jS-strands, and the chains align in a parallel stack, constrained by the interchain disulfide bonds. One pair of stacked /i-stran ds is curved... 

Grimes, J. M., et al. (1997). An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy. Structure 5,885-893. 

Stansfield 1, Jones KM, Kushnirov VV, Dagkesamanskaya AR, Poznyakovski Al, Paushkin SV, Nierras CR, Cox BS, Ter-Avanesyan MD, Tuite ME (1995) The products of the SUP45 (eRFl) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J 14 4365 373 Stansfield 1, Eurwilaichitr L, Akhmaloka, Tuite ME (1996) Depletion in the levels of the release factor eRFl causes a reduction in the efficiency of translation termination in yeast. Mol Microbiol 20 1135-1143 Stansfield 1, Kushnirov VV, Jones KM, Tuite ME (1997) A conditional-lethal translation termination defect in a sup45 mutant of the yeast Saccharomyces cerevisiae. Fur J Biochem 245 557-563 Stark H (2002) Three-dimensional electron cryomicroscopy of ribosomes. Curr Protein Pept Sci 3 79-91... 

Bottcher, B., Lucken, U., and Graber, P. (1995). The structure of the H+-ATPase from chloroplasts by electron cryomicroscopy. Biochem. Soc. Trans., 23, 780-5. 

Yang, Z., Kollman, J. M., Pandi, L., and Doolittle, R. F. (2001). Crystal structure of native chicken fibrinogen at 2.7 A resolution. Biochemistry 40, 12515-12523. Yonekura, K., Maki-Yonekura, S., and Namba, K. (2003). Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature 424, 643-650. Zhang, L., and Hermans, J. (1993). Calculation of the pitch of the a-helical coiled coil An addendum. Proteins 17, 217-218. 

Suzuki, H., Yonekura, K., and Namba, K. (2004). Structure of the rotor of the bacterial flagellar motor revealed by electron cryomicroscopy and single-particle image analysis./. Mol. Biol. 337, 105-113. 

Hirose, K., Lowe,J., Alonso, M., Cross, R. A., and Amos, L. A. (1999). Congruent docking of dimeric kinesin and ncd into three-dimensional electron cryomicroscopy maps of microtubule-motor ADP complexes. Mol. Biol. CeU 10, 2063-2074. 

Kozielski, F., Amal, I., and Wade, R. H. (1998). A model of the microtubule-kinesin complex based on electron cryomicroscopy and X-ray crystallograhpy. Curr Biol. 8, 191-198. 

K. H. (1990). Model for the structure of bacteriorhodopsin based on high-resolution electron cryomicroscopy./. Mol. Biol. 213, 899-929. 

Akiba, T., Toyoshima, C., Matsunaga, T., Kawamoto, M., Kubota, T., Fukuyama, K., Namba, K., and Matsubara, H., 1996, Three-dimensional structure of bovine cytochrome bcl complex by electron cryomicroscopy and helical image reconstruction. Nature Struct. Biol. 3 553n561. 

K. Yonekiua, S. Maki-Yonekura, K. Namba, Complete Atomic Model of the Bacterial Flagellar Filament by Electron Cryomicroscopy , Nature, 424, 643 (2003)... 

Grimes et al., 1998 Reinisch et al, 2000 Wikoff et at, 2000). Likewise, advances in electron cryomicroscopy and image reconstruction techniques allow time-resolved investigations of structural transitions associated with capsid assembly and maturation (Conway et al., 2001 Lawton et al, 1997). These developments have been paralleled by refinements in the molecular approaches used for sample preparation, with the result that synthesis of assembly intermediates and end products has become routine for many viruses. 

DETERMINATION OF ICOSAHEDRAL VIRUS STRUCTURES BY ELECTRON CRYOMICROSCOPY AT SUBNANOMETER RESOLUTION... 

X-Ray crystallography is the method of choice for revealing atomic structures of large macromolecules and viruses. As shown in various examples in this volume, electron cryomicroscopy has emerged rapidly and has become a parallel technique to reveal additional information about virus structures, even in the situation in which the crystal structure of the virus may have already been obtained. The information that can be extracted from a hybrid approach of X-ray crystallography and electron cryomicroscopy is... 

For more complex structures, it is possible to combine electron cryomicroscopy structures with sequence-based secondary structure predictions to interpret the observed secondary structure elements. In the outer shell protein P8 of rice dwarf virus (RDV), where nine helices were predicted in the domain formed by the N and C termini, it was possible to match the lengths of the helices identified in the 3-D density map to those predicted from a consensus secondary structure analyses (Fig. 13a see Color Insert). The connections between the helical densities can be seen in the lower domain of P8, allowing us to establish a rough backbone model for the lower domain of P8 (Zhou et at, 2001). 

Bottcher, B., Kiselev, N. A., Stel Mashchuk, V. Y., Perevozchikova, N. A., Borisov, A. V., and Crowther, R. A. (1997a). Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy. / Virol. 71, 325-330. 

Chiu, W., McGough, A., Sherman, M. B., and Schmid, M. F. (1999). High-resolution electron cryomicroscopy of macromolecular assemblies. Trends Cell Biol. 9, 154-159. ... 

Prasad, B. V. V., and Estes, M. K. (2000). Electron Cryomicroscopy and Computer Image Processing Techniques Use in Structure-Function Studies of Rotavirus. Humana Press, Totowa, NJ. 

Zhou, Z. H., Baker, M. L., Jiang, W., Dougherty, M., Jakana, J., Dong, G., Lu, G., and Chiu, W. (2001). Electron cryomicroscopy and bioinformatics suggest protein fold models for rice dwarf virus. Nat. Struct. Biol. 8, 868-873. 

It is possible that the structure of HA2 in its low pH-converted form represents the results of multiple, sequential conformational changes (White and Wilson, 1987 Korte et al, 1999), and that, under certain conditions, intermediate states can be trapped. The first intermediate is likely to be one in which the fusion peptides have emerged from their buried positions. An electron cryomicroscopy reconstruction of HA exposed to low pH for short time periods at 4°C revealed relatively... 

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