Cryo-electron tomography of individual protein molecules

Abstract: Averaging methods of determining structure, such as X-ray diffraction, do not preserve information about the flexibility of molecules. Cryo-electron tomography allows us to reconstruct individual hydrated objects. The method is limited to low-resolution, but it can be used to study dynamic structures, such as very large macromolecular complexes, and to perform in situ analysis of cellular organelles. This thesis explores the expansion of the cryo-electron tomography method to individual protein molecules. Tomographic structures of four proteins, ranging in size from 90 to 150 kDa, are presented in the current thesis. We have analysed the structure and flexibility of the antibody immunoglobulin G (IgG). The tomograms reveal y-shaped IgG molecules with three protruding subunits. We show that the tomographic structures are consistent with X-ray crystallographic structures of IgG and that the three 50 kDa subunits were resolved with accuracy. Each subunit has a similar structure in the tomograms and in the Xray map. Notably, the positions of the subunits differed greatly from one molecule to another. The large flexibility of IgG in solution is most likely of functional significance in antigen recognition. We have investigated a larger number of individual IgG molecules, measured equilibrium distribution of the molecule in terms of the relevant angular coordinates and built a model of the dynamics of IgG in solution. The hepatocyte growth factor/scatter factor (HGF/SF) controls the growth, morphogenesis or migration of epithelial, endothelial and muscle progenitor cells. We have defined the main conformations of inactive single-chain HGF/SF and active two-chain HGF/SF. Furthermore we present structures of the receptor tyrosine kinase MET and of MET bound to two-chain HGF/SF. These structures reveal the mechanism of HGF/SF activation and clarify the mode of binding to MET. Nuclear receptors play important roles in development and tissue homeostasis, and have been implicated in many disease states. We present the structure of the full-length Glucocorticoid Receptor (GR) protein, activated by a synthetic hormone agonist. Three asymmetric domains are clearly defined in the structure of the GR monomer, and two low-density regions, interpreted as hinge regions, connect the domains. The three domains were further characterized by multi-resolution docking procedures and by visualizing GR in complex with a monoclonal antibody. These studies show that cryo-electron tomography can be used to visualize individual proteins molecules with a molecular weight below 200 kDa. Thus, the method can be applied to flexible multi-domain proteins that have not been amenable to high-resolution methods of determining structure.