EM Analysis of Protein Structure


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. 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. pombeAPC/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., with permission from Elsevier. http://www.sciencedirect.com/science/journal/10972765.



Adrian M, Dubochet J, Fuller SD and Harris JR (1998) Cryo‐negative staining. Micron 29: 145–160.

Agrawal RK and Frank J (1999) Structural studies of the translational apparatus. Current Opinion in Structural Biology 9: 215–221.

Bartolome S, Bermudez A and Daban JR (1994) Internal structure of the 30 nm chromatin fiber. Journal of Cell Science 107(part 11) : 2983–2992.

Bremer A, Henn C, Engel A, Baumeister W and Aebi U (1992) Has negative staining still a place in biomacromolecular electron microscopy? Ultramicroscopy 46: 85–111.

Cheng Y, Zak O, Aisen P, Harrison SC and Walz T (2004) Structure of the human transferrin receptor‐transferrin complex. Cell 116: 565–576.

Crampton DJ, Ohi M, Qimron U, Walz T and Richardson CC (2006) Oligomeric states of bacteriophage T7 gene 4 primase/helicase. Journal of Molecular Biology 360: 667–677.

Frank J (2001) Cryo‐electron microscopy as an investigative tool: the ribosome as an example. BioEssays 23: 725–732.

Frank J and Radermacher M (1992) Three‐dimensional reconstruction of single particles negatively stained or in vitreous ice. Ultramicroscopy 46: 241–262.

Gieffers C, Dube P, Harris JR, Stark H and Peters JM (2001) Three‐dimensional structure of the anaphase‐promoting complex. Molecular Cell 7: 907–913.

Jurica MS, Sousa D, Moore MJ and Grigorieff N (2004) Three‐dimensional structure of C complex spliceosomes by electron microscopy. Nature Structural & Molecular Biology 11: 265–269.

Kastner B, Fischer N, Golas MM et al. (2008) GraFix: sample preparation for single‐particle electron cryomicroscopy. Nature Methods 5: 53–55.

Nangaku M, Sato‐Yoshitake R, Okada Y et al. (1994) KIF1B, a novel microtubule plus end‐directed monomeric motor protein for transport of mitochondria. Cell 79: 1209–1220.

Ohi MD, Feoktistova A, Ren L et al. (2007a) Structural organization of the anaphase‐promoting complex bound to the mitotic activator slp1. Molecular Cell 28: 871–885.

Ohi MD, Ren L, Wall JS, Gould KL and Walz T (2007b) Structural characterization of the fission yeast U5.U2/U6 spliceosome complex. Proceedings of the National Academy of Sciences of the USA 104: 3195–3200.

Ohi MD, Vander Kooi CW, Rosenberg JA et al. (2005) Structural and functional analysis of essential pre‐mRNA splicing factor Prp19p. Molecular and Cellular Biology 25: 451–460.

Sander B, Golas MM, Makarov EM et al. (2006) Organization of core spliceosomal components U5 snRNA loop I and U4/U6 Di‐snRNP within U4/U6.U5 Tri‐snRNP as revealed by electron cryomicroscopy. Molecular Cell 24: 267–278.

Sherratt MJ, Meadows RS, Graham HK, Kielty CM and Holmes DF (2009) ECM macromolecules: rotary shadowing and transmission electron microscopy. Methods in Molecular Biology (Clifton, NJ) 522: 175–181.

Stark H, Dube P, Luhrmann R and Kastner B (2001) Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle. Nature 409: 539–542.

Takagi J, Petre BM, Walz T and Springer TA (2002) Global conformational rearrangements in integrin extracellular domains in outside‐in and inside‐out signaling. Cell 110: 599–611.

Takagi J, Strokovich K, Springer TA and Walz T (2003) Structure of integrin alpha5beta1 in complex with fibronectin. EMBO Journal 22: 4607–4615.

van Heel M, Harauz G, Orlova EV, Schmidt R and Schatz M (1996) A new generation of the IMAGIC image processing system. Journal of Structural Biology 116: 17–24.

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.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Ohi, Melanie D(Dec 2009) EM Analysis of Protein Structure. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021885]