Cryomicroscopy

Hsu et al. [1.121] observed recrystallization on the recombinant CD4-IgG with a cry-omicroscope cooled to -60 °C by a cascade of four Peltier modules. The observation cell can also be evacuated for freeze-drying studies. 

Cryomicroscope studies have the advantage of showing pictures of the structural changes and the frozen product can be freeze-dried in most instruments. The pro duct layer is very thin and the product is quickly frozen. The behavior of the product during warming and drying therefore corresponds exactly only to a quickly frozen product. To simulate a thermal treatment is difficult because of the thin layer. However, experience shows, that critical temperatures taken from such studies are valuable, especially if they are supported by e.g. ER data of a more slowely frozen product. 

A cryomicroscope that permits quantitative evaluation of the pictures was described by Cosman et al. [1.28], The unit has four distinctive features  

The volume of oocytes of a rhesus monkey placed in 10% v/v dimethyl sulfoxide (DMSO) is reduced to almost one third, since the water can diffuse out of the cell into the surrounding, but the DMSO cannot enter the cell during the same time (measured at 23 °C). 

Cryo-SEM and cryo-TEM are the methods that have been developed for study of polymers at low temperatures, and these will be discussed in this section. Low temperature stages for AFM are just becoming available. There is some published work on PDES (polydiethylsi-loxane) materials obtained in an AFM cooled to -50°C using thermoelectric cooling [588]. 

Dudler, M.C. Grob and D. Merian, Polym. Degrad. Stab. 68 (2000) 373. 

Hearle, J.T. Sparrow and P.M. Cross, The Use of the Scanning Electron Microscope (Pergamon, Oxford, 1972). 

Goodhew, in Practical Methods in Electron Microscopy, Vol. 1, edited by A.M. Glauert 

Castano, A. Alvarez-Castillo, G. Vazquez-Polo, D. Acosta and V. Gonzalez, Microsc. Res. Tech. 40 (1998) 41. 

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Bdttcher B, Wynne S A and Crowther R A 1997 Determination of the foid of the core protein of hepatitis B virus by eiectron cryomicroscopy Nature 386 88-91... 

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. 

One technique that does probe the foam stmcture directly is cryomicroscopy. The foam is rapidly frozen, and the soHd stmcture is cut open and imaged with an optical or electron microscope (14). Such methods are widely appHcable and provide a direct image of the foam stmcture however, they destroy the sample and may also perturb the foam stmcture in an uncontroUed manner during the freezing. 

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. 

There are other variations of these imbibition methods, plus somewhat more direct methods such as cryomicroscopy (to visualize the distribution of liquids within the pores), and NMR (which is based on the different proton relaxation rates that occur for water near oil-wet versus water-wet surfaces). 

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... 

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