De meeste directe manier om structuren van moleculen in onze cellen te bestuderen is door ze sterk vergroot te bekijken. In dit project zal zo met een elektronenmicroscoop
gekeken worden naar mitochondriën, de energiefabrieken van onze cellen. De onderzoeksresultaten kunnen ons meer leren over de moleculaire oorzaken van veroudering.

The Endoplasmic Reticulum (ER) membrane in all eukaryotic cells has an intricate protein network that facilitates protein biogenesis and homeostasis. The molecular complexity and sophisticated regulation of this machinery favours studying it in its native microenvironment by novel approaches. Cryo-electron tomography (CET) allows 3D imaging of membrane-associated complexes in their native surrounding. Computational analysis of many subtomograms 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 biogenesis 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 substrates such as 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.

The aim of this proposal is to develop new computational methodology to enable high-resolution insights into macromolecular complexes from cryo-electron tomographic data. In particular, the proposed algorithms will be key to molecular structural biology in situ, i.e. to study macromolecular complexes in their native settings. The specific aims are:
1. Determination and correction of local beam-induced motion for subtomogram analysis
2. A constrained, regularized Fourier-based iterative tomographic reconstruction algorithm
3. Integration of advanced tomographic reconstruction into subtomogram analysis
4. Documentation of algorithms and appropriate tutorials