Structure and function of large macromolecular assemblies
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. Structure of the nuclear pore complex. Membranes are coloured in grey, the scaffold structure in yellow and the nuclear basket in transparent brown.
Research in the Beck group combines biochemical approaches, proteomics and cryo-electron microscopy to study large macromolecular assemblies.
Previous and current research
Cryo-electron tomography is the ideal tool to observe molecular machines at work in their native environment (figure 1). In combination with single particle analysis and averaging techniques, the overall structure of macromolecular assemblies can be determined (figure 2). Since the attainable resolution of the resulting 3D maps is moderate, the challenge ahead is to integrate information provided by complementary techniques and, in particular, to bridge the resolution gap towards high-resolution techniques (such as NMR and X-ray crystallography).
Proteomics 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 within macromolecular assemblies. Together with cross-linking techniques, the protein interfaces are revealed. The spatial information obtained in this way facilitates the fitting of high resolution structures into cryo-EM maps in order to build atomic models of entire molecular machines.
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 can occur in several copies within an individual assembly. Studying their structure and function is a challenging task, not only due to their compositional complexity, but also because of their sheer size that, in many cases, makes them inaccessible to biochemical purification.
We believe that the overall structure of intricate megadalton complexes can be elucidated through i) studying isolated protein subcomplexes that make up individual building blocks; and ii) understanding the stepwise assembly or disassembly process.
Future projects and goals
Our goals are:
- To develop integrated workflows for structure determination of large macromolecular assemblies such as the nuclear pore complex (figure 2).
- To study their function by imaging them in action.
- To reveal individual steps of their assembly and disassembly processes.