EM Analysis of Protein Structure

Melanie D Ohi, Vanderbilt University Medical School, Nashville, Tennessee, USA

Published online: December 2009

DOI: 10.1002/9780470015902.a0021885

Full Article on Wiley Online Library

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.

Keywords: vitrification; negative stain; metal shadowing; single-particle EM; structural biology

	Figure 1. Preparing biological samples for EM analysis using negative staining, rotary shadowing or cryo-EM. (a) For negative staining, the sample is first absorbed onto a carbon-coated grid, washed in a heavy metal stain and then dried. Samples prepared using conventional negative staining generate high-contrast images in the electron microscope, but the particles contain severe structural deformations. (b) For rotary shadowing, the sample is absorbed on a grid and then slowly rotated while a thin layer of metal is deposited over the sample. This technique creates a thin metal shell that preserves the topological features of a specimen. (c) For cryo-EM, the sample is placed on a holey carbon grid, blotted and then rapidly plunged into a cryogen, resulting in particles trapped in vitrified ice. Samples prepared for cryo-EM are trapped in a vitrified ice, making it possible to generate high-resolution 3D maps.
	Figure 2. Structural characterization of the Prp19 tetramer using negative stain. Electron micrograph and representative projection averages of negatively stained His₆–Prp19. Upper panel shows a typical micrograph area of negatively stained His₆–Prp19. Bar, 50 nm. The middle panel shows 12 representative averages of His₆–Prp19 particles. Side length of the average images is 40 nm. Reproduced from Ohi et al. (2005) with permission of the American Society for Microbiology.
	Figure 3. Comparing images of the Schizosaccharomyces pombe U5.U2/U6 spliceosomal complex in negative stain and vitrified ice. (a) Area of a typical electron micrograph of negatively stained U5.U2/U6 particles. Scale bar represents 100 nm. (b) A typical electron micrograph area of U5.U2/U6 particles in vitrified ice. Scale bar represents 100 nm.
	Figure 4. 3D reconstruction of the S. pombe APC/C. Single-particle cryo-EM was used to determine the 27 Å 3D structure of the APC/C. The complex is tilted about its vertical axis by 90°. The APC/C has a tricorn shape with a large central cavity and a prominent horn. Scale bar represents 5 nm. Reprinted from Ohi et al. (2007a), with permission from Elsevier. http://www.sciencedirect.com/science/journal/10972765.

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Further Reading
	Frank J (2002) Single-particle imaging of macromolecules by cryo-electron microscopy. Annual Review of Biophysics and Biomolecular Structure 31: 303–319.
	Leschziner AE and Nogales E (2007) Visualizing flexibility at molecular resolution: analysis of heterogeneity in single-particle electron microscopy reconstructions. Annual Review of Biophysics and Biomolecular Structure 36: 43–62.
	Ohi M, Li Y, Cheng Y and Walz T (2004) Negative staining and image classification – powerful tools in modern electron microscopy. Biological Procedures Online 6: 23–34.

Article Title:	EM Analysis of Protein Structure
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EM Analysis of Protein Structure

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