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TRIPEPTIDYL PEPTIDASE II; 26S PROTEASOME; TOMOGRAPHY
Abstract:
Today's biomolecular electron microscopy uses essentially three different imaging modalities: (i) electron crystallography, (ii) single particle analysis and (iii) electron tomography. Ideally, these imaging modalities are applied to frozen-hydrated samples to ensure an optimum preservation of the structures under scrutiny. Electron crystallography requires the existence of two-dimensional crystals. In principle, electron crystallography is a high-resolution technique and it has indeed been demonstrated in a number of cases that near-atomic resolution can be attained. Single-particle analysis is particularly suited for structural studies of large macromolecular complexes. The amount of material needed is minute and some degree of heterogeneity is tolerable since image classification can be used for further 'purification in silico'. In principle, single particle analysis can attain high-resolution but, in practice, this often remains an elusive goal. However, since medium resolution structures can be obtained relatively easily, it often provides an excellent basis for hybrid approaches in which high-resolution structures of components are integrated into the medium resolution structures of the holocomplexes. Electron tomography can be applied to non-repetitive structures. Most supramolecuar structures inside cells fall into this category. In order to obtain three-dimensional structures of objects with unique topologies it is necessary to obtain different views by physical tilting. The challenge is to obtain large numbers of projection images covering as wide a tilt range as possible and, at the same time, to minimize the cumulative electron dose. Cryoelectron tomography provides medium resolution three-dimensional images of a wide range of biological structures from isolated supramolecular assemblies to organelles and cells. It allows the visualization of molecular machines in their functional environment (in situ) and the mapping of entire molecular landscapes.
