Structure cryomicroscopy

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. 

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. 

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. 

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. 

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. 

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... 

Baker, M. L. (2002). Development and Applications of Intermediate Resolution Structural Analysis Tools Integrating Bioinformatics and Electron Cryomicroscopy. Ph.D. thesis. Baylor College of Medicine, Houston, TX. 

Zhou, Z. H., Prasad, B. V. V., Jakana, J., Rixon, F. J., and Chiu, W. (1994). Protein subunit structures in the herpes simplex virus A-capsid determined from 400kV spot-scan electron cryomicroscopy./. Mol. Biol. 242, 456-469. 

Johnson, Fig. 3. A composite of the capsid of paricoto virus (a nodavirus related to flock house virus) produced by electron cryomicroscopy and the packaged nucleic acid produced from the X-ray structure. The regions of the capsid protein that interact with the RNA are also shown from the X-ray structure (Tang et al, 2001). 

Orlova EV, Sherman MB, Chiu W, Mowri H, Smith LC, Gotto AM. Jr. Three-dimensional structure of low density lipoproteins by electron cryomicroscopy. Proc. Natl. Acad. Sci. U.S.A. 1999 96 8420-8425. 

Many medium resolution structures of macromolecular assemblies (e.g., ribosomes), spherical and helical viruses, and larger protein molecules have now been determined by electron cryomicroscopy in ice. Four atomic resolution structures have been obtained by electron cryomicroscopy of thin 2D crystals embedded in glucose, trehalose, or tannic acid (11-14), where specimen cooling reduced the effect of radiation damage. One of these, the structure of bacteriorhodopsin (1 l)provided the first structure of a seven-helix membrane protein. The medium resolution density distributions can often be interpreted in terms of the chemistry of the structure if a high resolution model of one or more of the component pieces has already been obtained by X-ray, electron microscopy, or NMR methods. As a result, the use of electron microscopy is becoming a powerful technique for which, in some cases, no alternative approach is possible. Useful reviews [e.g., Dubochet et al. (9), Amos et al. (15), Walz and Grigorieff (16), and Baker et al. (17)] and a book [Frank (18)] have been written. 

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