Integrative structural biology of ER protein biogenesis and quality control
Longtime strengths of the group are three-dimensional (3D) cryoelectron microscopy (cryo-EM), innovation in 3D image processing, and computational modeling of macromolecular assemblies. These cornerstones enable us to pursue an integrative approach to structural characterization of assembly function, which is particularly powerful to study weakly- and membrane-associated assemblies that are inaccessible to many other methods. We are primarily interested in the structural basis of synthesis, folding, post-translational modification, and degradation of secretory pathway proteins, which constitute approximately one third of the proteome of most eukaryotic cells.
Cryoelectron tomography (CET) is a versatile imaging modality that allows structural analysis of macromolecular complexes in their physiological microenvironment, e.g., in whole cells, purified organelles or lysates. Since the resolution of a cryo-tomogram is limited by the applicable electron dose, averaging subtomograms containing identical types of macromolecules can provide much higher resolution insights than a single tomogram (Fig. 1A). Our broad goal is to further improve resolution of subtomogram averages, to enable most accurate disentanglement of different conformational states in situ, and to build atomic models that explain the observed data.
In mammalian cells secretory pathway proteins are typically co-translationally translocated across the ER membrane. The ER-translocon is an integral membrane protein complex comprising the protein-conducting channel Sec61 and several associated complexes involved in post-translational processing (e.g., glycosylation, signal peptide cleavage, and folding). We aim to study the structural details of co-translational translocation and concomitant processing in situ.
ER-residing proteins are sensitive to defects in glycosylation or disulphide bridge formation. Two mechanisms have evolved to respond to ER stress, ER associated degradation (ERAD) and the unfolded protein response (UPR), both implicated in disorders such as Alzheimer’s and Parkinson’s disease. We aim to image these structurally elusive processes at the molecular level using CET.
Image: Subtomogram analysis applied to the ER-associated mammalian ribosome. A: Principle of subtomogram analysis. A tomogram of a ribosome-studded microsome is reconstructed from projections. Particles (ribosomes) are detected using automated methods and the corresponding subtomograms are aligned. Classification allows resolving different populations of particles. B: Architecture of the major complexes constituting the native translocon (protein-conducting channel Sec61, TRAP complex, and oligosaccharyl transferase complex, OST) resolved by subtomogram averaging to ~8 Å.