Figure 1: Cryo-EM: High-resolution helical reconstruction of tobacco mosaic virus at 3.3 Å resolution using single-particle cryo-EM from direct electron detectors. Top: helical rod superimposed on cryomicrograph. Center: cross section comprising 17 subunits. Bottom: close-up of a-helix including sidechain density.
The Sachse group uses electron cryomicroscopy to study the structures of autophagy complexes, with the aim of elucidating the mechanisms by which cells eliminate aberrant structures such as large protein aggregates.
Previous and current research
Autophagy (from the Greek, meaning ‘to eat oneself’) is the cell’s housekeeping mechanism to engulf and degrade long-lived proteins, macromolecular aggregates, damaged organelles and even microbes in double-membrane vesicles called autophagosomes. In our group, we investigate the molecular structures involved in autophagy as they provide fundamental insights for our understanding of aberrant cellular processes like cancer, ageing or infection.
We study the structures of molecular assemblies using biochemical and biophysical techniques, and subsequently visualise them by electron cryomicroscopy (cryo-EM). By this technique, large macromolecular structures and multi-protein complexes can be studied in their near-native environment without the need for crystallisation. Small amounts of material are sufficient to obtain ‘snapshots’ of single particles in the electron cryomicroscope. The molecular images are combined by computer-aided image processing techniques to compute their 3D structures. As recent advances in hardware and software have led to a wave of atomic-resolution structures, cryo-EM shows great promise in becoming a routine tool for high-resolution structure determination of large macromolecules. To further realise the potential of the technique, the scientific community is still in great need of hardware-based improvements and software enhancements. Therefore, we are also interested in developing techniques, including sample preparation and data processing, to routinely achieve atomic-resolution structures by single-particle cryo-EM. For example, in our group we actively develop the software SPRING for high-resolution cryo-EM structure determination of specimens with helical symmetry.
Figure 2: Autophagy: A de novo double membrane vesicle entraps large cytosolic cargo such as macromolecules, organelles, protein aggregates and even pathogens destined for degradation in the lysosome.
Future projects and goals
Multiprotein complexes are essential mediators in the events leading to autophagy. On the structural level however, little is known about their 3D architecture. Fundamental questions on the nature of these complexes need to be addressed:
- How are protein deposits structurally linked to autophagy?
- What are the shapes of these multiprotein assemblies at the membrane?
- How do they give rise to the cellular structure of the autophagosome?
Spring is a single-particle based helical reconstruction package and has been used to determine 3D structures of a variety of highly ordered and less ordered specimens from electron micrographs.