Three-dimensional (3-D) image reconstruction of electron microscopy (EM) data offers a powerful tool in structural biology that provides important information about the structure and function of individual biomacromolecules, their oligomers, and supramolecular assemblies. Forming an important link between atomic-resolution X-ray or NMR data of individual proteins and their supramolecular complexes, 3-D EM data allow the combining of structural information from all sources to assess protein function at atomic detail. Within the last several years, a range of image reconstruction methods were introduced aiming to determine 3-D structures of biomacromolecular assemblies to intermediate resolution (i.e., ≤20 Å). To achieve a faithful representation of the specimen under investigation its preparation for EM is of critical importance. Whereas earlier 3-D reconstructions were predominantly obtained with negatively stained specimens, more recently the potential of cryofixation/ice embedding has definitely become widely recognized. Imaging the biomacromolecule itself rather than a heavy metal cast surrounding it, by freezing the sample in its physiological buffer environment, cryofixation/ice embedding was found to reduce adsorption artifacts and to reproduce certain structural details more accurately and faithfully. Most importantly, in addition to mapping out the overall size and shape of biomacromolecules, cryo-EM enables us to look “inside” biomacromolecules and image their secondary structure elements or even atomic detail. Despite these obvious advantages of ice embedding over negative staining, however, both preparation methods have their own strengths and limitations which should be carefully evaluated based on the particular structural and biological question asked.
Journal of Structural Biology – Elsevier
Published: Sep 1, 1996
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