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Journal of electron microscopy technique

Comparisons of the low-resolution structures of ornithine decarboxylase by electron microscopy and X-ray crystallography: the utility of methylamine tungstate stain and Butvar support film in the study of macromolecules by transmission electron microscopy.


PMID 1715911

Abstract

The structure of ornithine decarboxylase (Mr approximately 1.04 x 10(6] from Lactobacillus 30a was investigated by electron microscopy and x-ray crystallography. Electron micrographs showed the structure to be well preserved in methylamine tungstate stain. The molecules interacted little with the Butvar support film, yielding three unique projections: a hexagonal ring (front view) and two rod-shaped projections (edge views). Stereo pairs revealed a novel feature of the Butvar film in that some molecules were suspended in the stain in random orientations. Consequently, the relatedness of the hexagonal ring and the rod-shaped particles could be demonstrated since some particle shapes interconverted when the stage was tilted +/- 45 degrees. The two edge views were related by a 30 degrees rotation about the sixfold axis. Image averaging of the three primary views suggested a dodecamer (point group symmetry 622) composed of two hexameric rings, apparently in an eclipsed configuration. To investigate the structural organization of the complex, the dissociation of the enzyme was studied by electron microscopy. The dissociation process involved the initial breakage of the ring followed by separation of dimers from the ring (one subunit from each of the two hexamers). Thus, the dodecamer forms as a hexamer of dimers rather than a dimer of hexamers. These structural studies were confirmed and extended by x-ray crystallographic analysis. A 4.0-A resolution electron density map revealed two hexameric rings, consisting of six closely associated dimers, tilted approximately 10 degrees with respect to the molecular twofold axis. Electron density projections of the three primary views of the molecule derived from the x-ray data corresponded closely to those obtained from image averaging of the electron microscopy data, thereby establishing in a novel way the reliability of the electron microscopy studies. Methylamine tungstate stain and Butvar support film therefore offer unique advantages for investigating protein structures by electron microscopy.

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