EM Analysis of Protein Structure

Abstract

Abstract Proteins carry out most cellular processes in the context of dynamic multiprotein assemblies. Although mapping the large number of protein interactions found within cells is progressing rapidly, we still lack knowledge about how proteins assemble into macromolecular machines to perform their functions. Electron microscopy can be used to determine the shape and structures of biological molecules that are difficult to study by more traditional structural approaches, such as X‐ray crystallography and NMR analysis. However, since biological molecules are composed mainly of low electron scattering atoms and contain large amounts of water, it is necessary to use special preparation techniques to protect the structural integrity of biological specimens in the vacuum of the electron microscope and improve the amount of contrast created by the sample. Negative staining, rotary shadowing and cryo‐EM are all powerful techniques that are useful for examining the structures and molecular organization of biological complexes by EM. Key concepts: Negative staining is a valuable approach to characterizing the structural homogeneity of a sample. Negative staining can be used to quantify major conformational shifts of proteins and complexes in response to the addition of inhibitors, activators or ligands. Rotary shadowing provides information about the surface topology of a specimen. Cryo‐EM preserves specimens in a near‐native environment and is used to generate high‐resolution 3D structures of large macromolecular complexes.