Figure 1. Cryo-electron tomogram of a fraction of the cytoplasm of a human cell. Microtubules are coloured in orange, stress fibres in grey, protein complexes in green, membranes in cyan and vesicular contents in yellow.
Figure 2. Model of the scaffold arrangement of the human Nuclear Pore Complex revealed by an integrated approach consisting of cryo-electron tomography, single particle EM, cross-linking MS and structural modelling (Bui, von Appen et al., Cell, 2013).
Research in the Beck group combines biochemical approaches, proteomics and cryo-electron microscopy to study large macromolecular assemblies.
Previous and current research
Integrated structure determination approaches: Research in our laboratory combines biochemical approaches, proteomics and cryo-electron microscopy to study the structure and function of large macromolecular assemblies. Cryo-electron tomography is the ideal tool to observe molecular machines at work in their native environment (figure 1). Since the attainable resolution of the tomograms is moderate, the challenge ahead is to integrate information provided by complementary approaches in order to bridge the resolution gap towards high-resolution techniques (NMR, X-ray crystallography). Mass spectrometry approaches can provide the auxiliary information that is necessary to tackle this challenge. Targeted mass spectrometry can handle complex protein mixtures and, in combination with heavy labelled reference peptides, provides quantitative information about protein stoichiometries. Using this together with cross-linking techniques can reveal protein interfaces. The spatial information obtained in this way facilitates the fitting of high-resolution structures into cryo-EM maps in order to build pseudo-atomic models of entire molecular machines (figure 2).
Large macromolecular assemblies: Megadalton protein complexes are involved in a number of fundamental cellular processes such as cell division, vesicular trafficking and nucleocytoplasmic exchange. In most cases such molecular machines consist of a multitude of different proteins that occur in several copies within an individual assembly. Their function is often fine-tuned towards context specific needs by compositional remodelling across different cell-types. Structural variations occur through stoichiometric changes, subunit switches or competing protein interfaces. Studying the structure and function of Megadalton protein complexes is a challenging task, not only due to their compositional complexity but also because of their sheer size, which makes them inaccessible to biochemical purification.
Future projects and goals
- To develop integrated workflows for structure determination of large macromolecular assemblies such as the nuclear pore complex (figure 2).
- To reveal the function of cell-type specific variations of macromolecular assemblies.