Objective The Endoplasmic Reticulum (ER) membrane in all eukaryotic cells has an intricate protein network that facilitates protein biogene-sis and homeostasis. The molecular complexity and sophisticated regulation of this machinery favours study-ing it in its native microenvironment by novel approaches. Cryo-electron tomography (CET) allows 3D im-aging of membrane-associated complexes in their native surrounding. Computational analysis of many sub-tomograms depicting the same type of macromolecule, a technology I pioneered, provides subnanometer resolution insights into different conformations of native complexes. I propose to leverage CET of cellular and cell-free systems to reveal the molecular details of ER protein bio-genesis and homeostasis. In detail, I will study: (a) The structure of the ER translocon, the dynamic gateway for import of nascent proteins into the ER and their maturation. The largest component is the oligosaccharyl transferase complex. (b) Cotranslational ER import, N-glycosylation, chaperone-mediated stabilization and folding as well as oligomerization of established model substrate such a major histocompatibility complex (MHC) class I and II complexes. (c) The degradation of misfolded ER-residing proteins by the cytosolic 26S proteasome using cytomegalovirus-induced depletion of MHC class I as a model system. (d) The structural changes of the ER-bound translation machinery upon ER stress through IRE1-mediated degradation of mRNA that is specific for ER-targeted proteins. (e) The improved ‘in silico purification’ of different states of native macromolecules by maximum likelihood subtomogram classification and its application to a-d.This project will be the blueprint for a new approach to structural biology of membrane-associated processes. It will contribute to our mechanistic understanding of viral immune evasion and glycosylation disorders as well as numerous diseases involving chronic ER stress including diabetes and neurodegenerative diseases. Fields of science natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsnatural sciencesphysical sciencesopticsmicroscopyelectron microscopymedical and health sciencesclinical medicineendocrinologydiabetesmedical and health sciencesbasic medicinephysiologyhomeostasisnatural sciencesbiological sciencesmolecular biologystructural biology Programme(s) H2020-EU.1.1. - EXCELLENT SCIENCE - European Research Council (ERC) Main Programme Topic(s) ERC-2016-COG - ERC Consolidator Grant Call for proposal ERC-2016-COG See other projects for this call Funding Scheme ERC-COG - Consolidator Grant Host institution UNIVERSITEIT UTRECHT Net EU contribution € 2 496 611,00 Address HEIDELBERGLAAN 8 3584 CS Utrecht Netherlands See on map Region West-Nederland Utrecht Utrecht Activity type Higher or Secondary Education Establishments Links Contact the organisation Opens in new window Website Opens in new window Participation in EU R&I programmes Opens in new window HORIZON collaboration network Opens in new window Total cost € 2 496 611,00 Beneficiaries (1) Sort alphabetically Sort by Net EU contribution Expand all Collapse all UNIVERSITEIT UTRECHT Netherlands Net EU contribution € 2 496 611,00 Address HEIDELBERGLAAN 8 3584 CS Utrecht See on map Region West-Nederland Utrecht Utrecht Activity type Higher or Secondary Education Establishments Links Contact the organisation Opens in new window Website Opens in new window Participation in EU R&I programmes Opens in new window HORIZON collaboration network Opens in new window Total cost € 2 496 611,00
