Abstract
Like X-ray crystallography and NMR, electron microscopy (EM) is now widely applied to determine the structure of proteins and their macromolecular complexes. Single-particle analysis (SPA), which reconstructs the three-dimensional (3D) structure of a protein from its EM images using image processing, has an advantage when the target molecule is difficult to crystallize or only a small amount of protein can be obtained. The technique is based on the theory that two-dimensional EM images of a protein contain sufficient information to reconstruct the original 3D structure. SPA was developed when this theory was applied to ribosomes and the coat protein of icosahedral or helical symmetrical viruses. Because SPA does not require protein crystallization, it is widely applicable to the analysis of solubilized membrane proteins or supermolecular complexes. It allows conformational changes undergone by proteins to be documented. Many other EM-based structural analysis techniques are available in addition to SPA. Electron tomography reconstructs the 3D structure of a protein complex or a cell from a series of images recorded by tilting the specimen in the EM column. Electron crystallography can yield the high-resolution structure of proteins crystallized in two dimensions in a lipid bilayer. Atmospheric scanning electron microscopy directly observes cells in aqueous solution and has realized high-throughput immuno-EM of cells without hydrophobic treatment. It can also visualize protein microcrystals in the crystallization buffer.
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De Rosier DJ, Klug A (1968) Reconstruction of three dimensional structures from electron micrographs. Nature 217:130–134
Frank J, Zhu J, Penczek P et al (1995) A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome. Nature 376:441–444
Stark H, Mueller F, Orlova EV et al (1995) The 70S Escherichia coli ribosome at 23 Å resolution: fitting the ribosomal RNA. Structure 3:815–821
Bottcher B, Wynne SA, Crowther RA (1997) Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature 386:88–91
van Heel M, Gowen B, Matadeen R et al (2000) Single-particle electron cryo-microscopy: towards atomic resolution. Q Rev Biophys 33:307–369
Frank J (2006) Three-dimensional electron microscopy of macromolecular assemblies: visualization of biological molecules in their native state. Three-dimensional electron microscopy of macromolecular assemblies: visualization of biological molecules in their native state. Oxford University Press, New York
Bartesaghi A, Merk A, Banerjee S et al (2013) Electron microscopy. 2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor. Science 348:1147–1151
Jiang J, Pentelute BL, Collier RJ, Zhou ZH (2015) Atomic structure of anthrax protective antigen pore elucidates toxin translocation. Nature 521:545–549
Paulsen CE, Armache JP, Gao Y, Cheng Y, Julius D (2015) Structure of the TRPA1 ion channel suggests regulatory mechanisms. Nature 520:511–517
Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14:673–681
Bremer A, Henn C, Engel A, Baumeister W, Aebi U (1992) Has negative staining still a place in biomacromolecular electron microscopy? Ultramicroscopy 461:85–111
Nishiyama H, Suga M, Ogura T et al (2010) Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film. J Struct Biol 169:438–449
Mio K, Ogura T, Kiyonaka S et al (2007) The TRPC3 channel has a large internal chamber surrounded by signal sensing antennas. J Mol Biol 367:373–383
Sato C, Sato M, Iwasaki A, Doi T, Engel A (1998) The sodium channel has four domains surrounding a central pore. J Struct Biol 121:314–325
Sato C, Ueno Y, Asai K et al (2001) The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities. Nature 409:1047–1051
Yuuki H, Hasunuma Y, Komazawa K et al (1996) A sensitive enzyme immunoassay specific for salmon calcitonin. Biomed Res 17:257–259
Ishikawa E, Yoshitake S, Imagawa M, Sumiyoshi A (1983) Preparation of monomeric Fab’-horseradish peroxidase conjugate using thiol groups in the hinge and its evaluation in enzyme immunoassay and immunohistochemical staining. Ann N Y Acad Sci 420:74–89
Brenner S, Horne RW (1959) A negative staining method for high resolution electron microscopy of viruses. Biochim Biophys Acta 34:103–110
Taylor KA, Glaeser RM (1976) Electron microscopy of frozen hydrated biological specimens. J Ultrastruct Res 55:448–456
Adrian M, Dubochet J, Lepault J, McDowall AW (1984) Cryo-electron microscopy of viruses. Nature 308:32–36
Fujiyoshi Y, Mizusaki T, Morikawa K et al (1991) Development of a superfluid-helium stage for high-resolution electron-microscopy. Ultramicroscopy 38:241–251
Henderson R (2004) Realizing the potential of electron cryo-microscopy. Q Rev Biophys 37:3–13
Jin L, Milazzo AC, Kleinfelder S et al (2008) Applications of direct detection device in transmission electron microscopy. J Struct Biol 161:352–358
Milazzo AC, Moldovan G, Lanman J et al (2010) Characterization of a direct detection device imaging camera for transmission electron microscopy. Ultramicroscopy 110:744–747
Li X, Mooney P, Zheng S et al (2013) Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10:584–590
Grigorieff N (2013) Direct detection pays off for electron cryo-microscopy. Elife 2:e00573
van Heel M, Harauz G, Orlova EV, Schmidt R, Schatz M (1996) A new generation of the IMAGIC image processing system. J Struct Biol 116:17–24
van Heel M, Portugal R, Rohou A et al (2011) Four-dimensional cryo electron microscopy at quasi atomic resolution: IMAGIC 4D. In: Arnold E, Himmel DM, Rossmann MG (eds) Crystallography of biological macromolecules. Wiley, New York, pp 624–628
Frank J, Radermacher M, Penczek P et al (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116:190–199
Shaikh TR, Gao H, Baxter WT et al (2008) SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nat Protoc 3:1941–1974
Ludtke SJ, Baldwin PR, Chiu W (1999) EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128:82–97
Tang G, Peng L, Baldwin PR et al (2007) EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157:38–46
Scheres SH, Nunez-Ramirez R, Sorzano CO, Carazo JM, Marabini R (2008) Image processing for electron microscopy single-particle analysis using XMIPP. Nat Protoc 3:977–990
Sorzano CO, Marabini R, Velazquez-Muriel J et al (2004) XMIPP: a new generation of an open-source image processing package for electron microscopy. J Struct Biol 148:194–204
Marabini R, Masegosa IM, San Martin MC et al (1996) Xmipp: an image processing package for electron microscopy. J Struct Biol 116:237–240
Yasunaga T, Wakabayashi T (1996) Extensible and object-oriented system Eos supplies a new environment for image analysis of electron micrographs of macromolecules. J Struct Biol 116:155–160
Grigorieff N (2007) FREALIGN: high-resolution refinement of single particle structures. J Struct Biol 157:117–125
Scheres SH (2012) RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180:519–530
Ogura T, Sato C (2004) Automatic particle pickup method using a neural network has high accuracy by applying an initial weight derived from eigenimages: a new reference free method for single-particle analysis. J Struct Biol 145:63–75
Kawata M, Sato C (2013) Multi-reference-based multiple alignment statistics enables accurate protein-particle pickup from noisy images. Microscopy 62:303–315
Ogura T, Iwasaki K, Sato C (2003) Topology representing network enables highly accurate classification of protein images taken by cryo electron-microscope without masking. J Struct Biol 143:185–200
Crowther RA (1971) Procedures for three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs. Philos Trans R Soc Lond B Biol Sci 261:221–230
van Heel M (1987) Angular reconstitution – a posteriori assignment of projection directions for 3-D reconstruction. Ultramicroscopy 21:111–123
Frank J, Goldfarb W, Eisenberg D, Baker TS (1978) Reconstruction of glutamine synthetase using computer averaging. Ultramicroscopy 3:283–290
Radermacher M, Wagenknecht T, Verschoor A, Frank J (1986) A new 3-D reconstruction scheme applied to the 50S ribosomal subunit of E. coli. J Microsc 141:RP1–RP2
Ogura T, Sato C (2006) A fully automatic 3D reconstruction method using simulated annealing enables accurate posterioric angular assignment of protein projections. J Struct Biol 156:371–386
Maruyama Y, Ogura T, Mio K et al (2007) Three-dimensional reconstruction using transmission electron microscopy reveals a swollen, bell-shaped structure of transient receptor potential melastatin type 2 cation channel. J Biol Chem 282:36961–36970
Mio K, Kubo Y, Ogura T et al (2008) The motor protein prestin is a bullet-shaped molecule with inner cavities. J Biol Chem 283:1137–1145
Rosenthal PB, Henderson R (2003) Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol Biol 333:721–745
Trabuco LG, Villa E, Mitra K, Frank J, Schulten K (2008) Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure 16:673–683
Brandt F, Carlson LA, Hartl FU, Baumeister W, Grunewald K (2010) The three-dimensional organization of polyribosomes in intact human cells. Mol Cell 39:560–569
Tani K, Mitsuma T, Hiroaki Y et al (2009) Mechanism of aquaporin-4′s fast and highly selective water conduction and proton exclusion. J Mol Biol 389:694–706
Baumeister W (2002) Electron tomography: towards visualizing the molecular organization of the cytoplasm. Curr Opin Struct Biol 12:679–684
Gonen T, Cheng Y, Sliz P et al (2005) Lipid-protein interactions in double-layered two-dimensional AQP0 crystals. Nature 438:633–638
Jap BK, Zulauf M, Scheybani T et al (1992) 2D crystallization: from art to science. Ultramicroscopy 46:45–84
Lanyi JK (2004) Bacteriorhodopsin. Annu Rev Physiol 66:665–688
Kimura Y, Vassylyev DG, Miyazawa A et al (1997) Surface of bacteriorhodopsin revealed by high-resolution electron crystallography. Nature 389:206–211
Subramaniam S, Henderson R (2000) Molecular mechanism of vectorial proton translocation by bacteriorhodopsin. Nature 406:653–657
Maruyama Y, Ebihara T, Nishiyama H, Suga M, Sato C (2012) Immuno EM-OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM). J Struct Biol 180:259–270
Maruyama Y, Ebihara T, Nishiyama H et al (2012) Direct observation of protein microcrystals in crystallization buffer by atmospheric scanning electron microscopy. Int J Mol Sci 13:10553–10567
Hirano K, Kinoshita T, Uemura T et al (2014) Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM. Ultramicroscopy 143:52–66
Memtily N, Okada T, Ebihara T et al (2015) Observation of tissues in open aqueous solution by atmospheric scanning electron microscopy: applicability to intraoperative cancer diagnosis. Int J Oncol 46:1872–1882
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas, Structural basis of cell-signaling complexes mediating signal perception, transduction, and responses, and by grants from CREST; from the Ministry of Education, Culture, Sports, Science, and Technology; from Canon; and from AIST.
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Mio, K., Sato, M., Sato, C. (2016). Structural Biology and Electron Microscopy. In: Senda, T., Maenaka, K. (eds) Advanced Methods in Structural Biology. Springer Protocols Handbooks. Springer, Tokyo. https://doi.org/10.1007/978-4-431-56030-2_15
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DOI: https://doi.org/10.1007/978-4-431-56030-2_15
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