Fidelity of structure representation in electron micrographs of negatively stained protein molecules.

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Abstract
The fidelity of structure representation in EM of negatively stained proteins was investigated by conducting a systematic evaluation of such micrographs in terms of a known molecular structure, solved by X-ray crystallography. Microcrystals of IgG Dob were used as specimens in this comparison between micrograph images, optimized by computer image processing and reference images derived computationally from the crystal structure. To an effective resolution of 2 nm, a remarkably good correlation was observed between the experimental images and their idealized counterparts, which are unaffected by those factors (electron irradiation and dehydration) that could be primarly responsible for perturbation of protein structure during EM. Separate structural features resolved in these micrographs do not generally correspond to specific components of individual molecules but arise instead from complex superpositions involving several overlapping molecules.