The emerging discipline of electron tomography provides new and unprecedented opportunities todetermine three-dimensional cellular architecture at resolutions of ~50A or better, i.e., potentially high enoughto identify individual macromolecules such as proteins in a 3-D volume of a cell289. Electron tomography isespecially applicable for the structural analysis of cellular organelles and other macromolecular assembliesthat are too heterogeneous to be investigated by NMR or X-ray crystallographic techniques or even electronmicroscope-based methods that involve averaging of multiple copies of the same object to improve signal-tonoiseratios. Electron tomography bridges the gap between high resolution structure determination of proteincomplexes by NMR or X-ray crystallographic techniques and single-particle living cell imaging by lightmicroscopy using fluorescent probes. It extends the resolution of cellular imaging by one to two orders ofmagnitude over what is currently achieved using light microscopy.The fundamental principles underlying electron tomography and the strategy to use back-projectionalgorithms to extract 3-D information from a series of 2-D images recorded at different orientations were clearlyarticulated nearly four decades ago290'291. However, this field has seen a significant burst of activity in the lastfive years, principally propelled by the availability of tools for automated data acquisition using moderncomputerized microscopes. Efforts at imaging complex assemblies at room temperature as well as cryogenictemperatures have rapidly begun to provide many new insights into the 3-D architecture of cells289'292.During the immediate post-entry stages in HIV pathogenesis, the viral particle or, more precisely, itscontents must traverse through the cytoplasm of the host cell prior to nuclear import of the pre-integrationcomplex (PIC). Identification and characterization of these cellular events and viral and host interactions arecentral to understanding the molecular events that occur during HIV pathogenesis. NMR or X-raycrystallographic methods will provide high resolution structures of the viral and host protein complexes, whileconfocal microscopy in combination with fluorescent-labeling techniques allows tracking of viral particles andassessing the approximate localization of individual proteins in living cells. However, advancement of electronmicroscopic techniques, particularly electron tomography, will be essential to obtain higher resolutionsnapshots of the molecular arrangement of multiprotein complexes in the context of the host cellular structureand to provide the structural information necessary to fill the gap between NMR or X-ray crystallographicstructural determination and live cell imaging. We, therefore, expect that our work to develop approaches for 3-D cellular imaging will have a direct impact on cellular and structural studies of HIV infection.
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