Negative stain electron microscopy studies of mammalian proteins crucial for cellular life and death decisions
By: by Amanda Jean Rice
Series: Collections of the Harvard University Archives
Published: 2010
Language: eng
Number of pages: 132
Description: Electron microscopy (EM) allows visualization of proteins and macromolecular complexes up to near-atomic resolution. Single-particle EM uses low signal-to-noise ratio images of a biological molecule to reconstruct its three-dimensional (3D) structure. It can thus be used to obtain detailed structural information of molecules not easily studied by the more traditional structural techniques of X-ray crystallography and NMR spectroscopy. While cryo-EM of vitrified specimens is the best way to visualize biological molecules, negative staining remains a valuable tool for electron microscopists. The much higher contrast of negatively stained specimens, which is due to the heavy metal salts of the stain, as compared to vitrified specimens greatly improves the reliability of alignment and classification procedures used in the processing of EM images, particularly for molecules too small or too heterogeneous to be studied by cryo-EM. Although the resolution of 3D reconstructions calculated from images of negatively stained samples is limited to approximately 20 &Aring;, the fast and easy negative stain EM approach can still provide valuable structural insights.

Here I describe four macromolecular assemblies investigated by negative stain EM. In Part 1 (Chapter 3), I discuss collaborative efforts to characterize protein-protein interactions between members of the tumor necrosis factor receptor (TNFR) family and their signaling partners, using a combination of negative stain EM, X-ray crystallography and mutagenesis studies. Our results provide new insights into interaction mechanisms utilized by proteins of this family of signal transducers. In Part 2 (Chapter 4), I describe collaborative attempts to visualize two integral membrane protein species embedded in "nanodiscs," small patches of phospholipid bilayer that are stabilized in solution by amphipathic membrane scaffolding proteins shielding their hydrophobic edges. Results from these studies show that nanodiscs are a promising tool to study membrane proteins by single-particle EM. Overall, the work presented here deepens our understanding of the functions of proteins involved in processes critical for both cellular survival and programmed cell death. It also shows that single-particle EM of negatively stained specimens can make significant contributions to studies of biomolecular structures and can complement information provided by other structural and biochemical methods. iv
Notes: "June 2010."

Thesis (Ph.D., Committee on Higher Degrees in Biophysics)--Harvard University, 2010.

Includes bibliographical references.