Cryo-electron microscopy and X-ray crystallography

Rao, S., Poojary, M., Elliott, B., Melanson, L., Oriel, B., and Cohen, C. (1991). Fibrinogen structure in projection at 18 A resolution. Electron density by co-ordinated cryo-electron microscopy and X-ray crystallography. J. Mol. Biol. 222, 89-98. 

Wang, P., Porta, C., Chen, Z., Baker, T. S., and Johnson, J. E. (1992). Identification of a Fab interaction site (footprint) on an icosahedral virus by cryo-electron microscopy and X-ray crystallography. Nature 355, 275-278. 

Trabuco, L. G., Villa, E., Schreiner, E., Harrison, C. B., 8c Schulten, K. (2009). Molecular dynamics flexible fitting A practical guide to combine cryo-electron microscopy and X-ray crystallography. Methods, 49,174. 

The low-resolution phases initially required may be obtained in a variety of ways, but frequently these depend on other imaging techniques outside of X-ray crystallography. These may include transmission electron microscopy, cryo-electron microscopy, or atomic force microscopy. Low-resolution phases are even more often obtained by placing the known structure of a closely related virus, or complex, in the correct disposition in the unit cell (determined by rotation and translation functions) and using its low resolution calculated phases. 

STM and AFM are the most direct techniques for structural analysis because image generation does not involve substantial amounts of mathematical processing in order to obtain an image to view. Of these two techniques, AFM now holds the most utility for the visualisation of biological macromolecules and ambient temperatures and even in buffer conditions. Compared with X-ray crystallography, AFM is obviously also a technique still in development rather than fully mature, like cryo-electron microscopy. Nevertheless, we believe that it is necessary... 

Figure 6 A comparison of the rendered surfaces of TEM reconstructions of the active RecA-DNA filament (left) and the inactive RecA-DNA filament (right) with the RecA crystal surface (center). The RecA crystal surface, obtained by X-ray crystallography, has been rendered at low resolution, to be comparable with the TEM reconstructions. Both the active and inactive filament reconstructions are from averages of frozen-hydrated specimens imaged with cryo-EM. (Reproduced with permission from Egelman EH and StasiakA (1993) Electron microscopy of RECA-DNA complexes Two different states, their functional significance and relation to the solved crystal structure. Micron 24 309-324.)...

The structures of proteins are the key data for understanding the functions of proteins. There are three main techniques for measuring the structures of proteins nuclear magnetic resonance (NMR), cryo-electron microscopy (cryo-EM) and protein crystallography (X-ray diffraction). The solved structures of macromolecules, including proteins, nucleic acids and their complexes, are deposited in the Protein Data Bank (PDB). Up to 23 June of 2009, a total of 58 414 structures of macromolecules had been released in the PDB, and these were mostly protein structures (Table 7.1). Among these structures, about 86% were determined by protein crystallography. 

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