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High-Resolution Structure Determination of the Nuclear Pore Complex with Cryo-Electron Tomography (NUCLEAR PORE COMPLEX)
Start date: Mar 2, 2009, End date: Mar 1, 2011 PROJECT  FINISHED 

"In eukaryotes, nuclear pore complexes (NPCs) are large assemblies that selectively transport cargoes across the nuclear envelope. Determination of the architecture of the NPC in molecular detail is central to understanding nuclear transport. Structural analysis of the NPC is a major challenge given its sheer size and its dynamic nature. Cryo-electron tomography (cryo-ET) has successfully been used to elucidate the overall architecture of the NPC, but substantial improvements in resolution are needed to use cryo-ET maps as a scaffold into which high-resolution structures of individual nucleoporins (Nups) or subdomains can be fitted reliably. We propose to achieve such goal by using a novel technique for cryo-sectioning of frozen hydrated biological samples, focused ion beam (FIB) milling, to obtain thin sections of nuclei in lifelike conditions, that will permit us to achieve cryo-ET maps of 2-3nm. Furthermore, we will use metallothionein tags, that nucleate gold clusters, in order to localize individual Nups with high-accuracy within the NPC map. These data, together with cross linking data from mass spectroscopy to determine distances between pairs of Nups, available crystallographic structures of single Nups, and cryo-EM data of subdomains, will be integrated into a high-resolution structure of the entire NPC using a proteomics approach."

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