Towards higher resolution and cellular imaging using cryo-electron tomography

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Studying the structure of macromolecular complexes is essential for understanding how they function. Furthermore, studying the structure of the macromolecular complexes within their cellular context provides the most reliable information about their molecular function. Cryo-electron tomography (cryo-ET) is a powerful imaging technique that provides three-dimensional information about cellular architecture devoid of chemical fixation and staining artifacts. Cryo-ET can reach ~10 nm resolution and up to 3 nm with subtomogram averaging in silico. The limiting factor in cryo-ET resolution is the electron dose-limiting radiation damage and thick specimen. This resolution gives the information about the overall shape of the proteins but is not able to provide any molecular mechanism. My research focus is to push the resolution of cryo-ET to sub-nanometer resolution using the microtubule doublet as the test sample. At such resolutions, secondary structures are clearly identifiable and consequently, atomic models of subunits can be docked into the experimental tomographic maps to gain novel insights into protein-protein interactions and molecular mechanism. My research will also push cryo-ET to work on thicker sample i.e. looking at part of the cells by improving the signal-to noise ratio using the phase plate, direct electron detector and improved acquisition scheme. This will provide us with a unique ability to open a window into the cell to look at cellular process as unprecedented resolution. Furthermore, to address the dynamic nature of the cellular process, my laboratory will develop correlative light-electron microscopy technique, which combines the power of cryo-ET to resolve ultrastructure with the specific identification power of fluorescence light microscopy to precisely localize proteins of interest in the context of their surroundings to yield insights into fundamental biological mechanisms. ***The test sample for our method development will be the eukaryotic cilia, which are present in almost all cells in human. We plan to obtain the sub-nanometer structure of the microtubule doublet from the cilia using cryo-ET and the in situ structure of the intraflagellar transport complex, which is involved in the ciliary assembly within intact cells using CLEM. The technological developments outlined here will bridge multi-scale resolutions from isolated macromolecular complexes to in vivo cellular structures. The technology will provide important insights into the inner workings of cells and how they are altered in disease states. Our high-resolution structure will shed light onto the fundamental principals behind the biophysical properties of microtubule doublet, and lead to the mechanism of its function and potentially new ideas in biomaterial research.**