CORRELATED IMAGING OF SUPRAMOLECULAR COMPLEXES ANDCELLULAR COMPARTMENTS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A continuing challenge to structural biologists is the identification and structural analyses of large macromolecular complexes and organelles, including multi-component combinations of proteins, membranes and/or nucleic acids, especially within the context of tissues and cells. We at NCMIR have coined and have popularized the term "mesoscale" (from the Greek "mesos", middle) as the imaging range we mine to obtain new knowledge about the structure and dynamics of the macromolecular aggregates that compose the functional complexes within cells, tissues and organs. These mesoscale structures are imaged with light microscopy and with more detail using electron microscopy at moderate resolutions, achievable with numerous preparatory methods, as opposed to high resolution, atomic-level, 3-D structural methods, such as nuclear magnetic resonance (NMR), X-ray crystallography and molecular microscopy (e.g. electron cryomicroscopy). In addition, along with the task of creating systems biology computational approaches to protein-protein interactions (the "interactomes"), one would ideally like to place macromolecular complexes within their cellular environments in order to fully understand their functional interactions ("visual proteomics"). At NCMIR, we focus on the visualization of cells and organelles, and the identification of molecules for correlative light and electron microscopy (LM and EM, respectively). In this core section, we use the new specific staining strategies (tetracysteine domains/biarsenical ligands; quantum dot labeling; chemicals that specifically stain certain protein, carbohydrate or lipids) we have developed for a variety of biological specimens. We improve methods for ultrastructural preservation of labile tissues and photoconverted material. Finally, we apply advanced data collection and 3D reconstruction methods to push the resolution of our imaging techniques in order to bridge the gap between light microscopic imaging of cellular dynamics and direct visualization of macromolecular structure. As part of the extension of our previous work, we are exploring new specimen geometries that will allow for 360 degree rotation and therefore, subsequent tomograms will have no missing cone of data, as presently is the custom for standard single axis tilt or double axis tilt data collection on slab-like specimens.