2007Forster Review

Citation

Forster F, Hegerl R. Structure Determination In Situ by Averaging of Tomograms. Methods Cell Biol. 2007;79:741-767.

Abstract

Averaging single particles that have been extracted from cryoelectron tomograms is a powerful method for visualizing macromolecular complexes in situ. Cryoelectron tomography (CET) is uniquely suited to obtaining three-dimensional (3D) insights into pleiomorphic objects such as cells or organelles. Frozen-hydrated specimens provide excellent preservation of biological material, and thus a cryoelectron tomogram depicts a faithful image of complexes in situ. However, the resolution of CET is limited by the applicable electron dose; this limitation can be overcome for macromolecules that occur in identical copies within one or many tomograms by coherently averaging them. If the differently oriented and positioned particles are brought into precise register, their weak individual signals are amplified, resulting in an average that is of higher resolution. Here we report on the image processing that is needed to align the single particles to a common coordinate system, both in theory and in practice. Our method iteratively optimizes a scoring function that adopts its maximum value when all particles are in register; the scoring function is a cross-correlation function that accounts for the “missing-wedge effect,” currently the most prominent imaging artifact in CET. Structural characterization by CET and subsequent averaging can be particularly fruitful for unraveling the architecture of membrane-bound complexes.

Links

Article: http://dx.doi.org/10.1016/S0091-679X(06)79029-X