Electron Tomography of Paracrystalline 2D Arrays

  • Hanspeter Winkler
  • Shenping Wu
  • Kenneth A. TaylorEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 955)


Paracrystalline arrays possess specific types of disorder that reduce the structural information as well as resolution when spatially averaged over repeating motifs. Electron tomography combined with motif classification and averaging can solve the heterogeneity problem and provide information on the structural elements that give rise to the disorder. This chapter describes procedures that would be used in a typical tomography application to identify and characterize a paracrystalline specimen. Particular emphasis is given to actively contracting insect flight muscle, a specimen with particularly difficult to characterize structural heterogeneity and 2D paracrystalline arrays of myosin-V, from which a particularly high resolution motif average was obtained. All aspects of the study are described including data collection, merging of micrographs to produce the tomogram, alignment to an invariant structural element, classification and averaging of heterogeneous structures, and reassembly of focused class averages into high signal-to-noise ratio representations of the original raw repeats. Particular emphasis is placed on limitations of the various processes to produce the final class averages.

Key words

Cryoelectron microscopy Electron microscope tomography Computer-assisted image processing Three-dimensional imaging 



The research on insect flight muscle was supported by NIH Grant GM30598, the myosin-V reconstruction under NIH grant AR47421, and protomo is being developed under NIH grant GM82948.


  1. 1.
    Liu J, Taylor DW, Krementsova EB, Trybus KM, Taylor KA (2006) Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography. Nature 442:208–211PubMedGoogle Scholar
  2. 2.
    Taylor KA, Tang J, Cheng Y, Winkler H (1997) The use of electron tomography for structural analysis of disordered protein arrays. J Struct Biol 120:372–386PubMedCrossRefGoogle Scholar
  3. 3.
    Wu S, Liu J, Reedy MC, Tregear RT, Winkler H, Franzini-Armstrong C et al (2010) Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions. PLoS One 5:e12643PubMedCrossRefGoogle Scholar
  4. 4.
    Hampton CM, Liu J, Taylor DW, DeRosier DJ, Taylor KA (2008) The 3D structure of villin as an unusual F-Actin crosslinker. Structure 16:1882–1891PubMedCrossRefGoogle Scholar
  5. 5.
    Zhu P, Liu J, Bess J Jr, Chertova E, Lifson JD, Grise H et al (2006) Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 441:847–852PubMedCrossRefGoogle Scholar
  6. 6.
    Zhu P, Winkler H, Chertova E, Taylor KA, Roux KH (2008) Cryoelectron tomography of HIV-1 envelope spikes: further evidence for tripod-like legs. PLoS Pathog 4:e1000203PubMedCrossRefGoogle Scholar
  7. 7.
    Hu G, Liu J, Taylor KA, Roux KH (2011) Structural comparison of HIV-1 envelope spikes with and without the V1/V2 loop. J Virol 85:2741–2750PubMedCrossRefGoogle Scholar
  8. 8.
    Ye F, Liu J, Winkler H, Taylor KA (2008) Integrin alpha IIb beta 3 in a membrane environment remains the same height after Mn2+ activation when observed by cryoelectron tomography. J Mol Biol 378:976–986PubMedCrossRefGoogle Scholar
  9. 9.
    Ye F, Hu G, Taylor D, Ratnikov B, Bobkov AA, McLean MA et al (2010) Recreation of the terminal events in physiological integrin activation. J Cell Biol 188:157–173PubMedCrossRefGoogle Scholar
  10. 10.
    Liu J, Lin T, Botkin DJ, McCrum E, Winkler H, Norris SJ (2009) Intact flagellar motor of Borrelia burgdorferi revealed by cryo-electron tomography: evidence for stator ring curvature and rotor/C-ring assembly flexion. J Bacteriol 191:5026–5036PubMedCrossRefGoogle Scholar
  11. 11.
    Frank J, van Heel M (1982) Correspondence analysis of aligned images of biological particles. J Mol Biol 161:134–137PubMedCrossRefGoogle Scholar
  12. 12.
    Van Heel M, Frank J (1981) Use of multivariate statistics in analysing the images of biological macromolecules. Ultramicroscopy 6:187–194PubMedGoogle Scholar
  13. 13.
    Winkler H, Taylor KA (2006) Accurate marker-free alignment with simultaneous geometry determination and reconstruction of tilt series in electron tomography. Ultramicroscopy 106:240–254PubMedCrossRefGoogle Scholar
  14. 14.
    Winkler H (2006) 3D reconstruction and processing of volumetric data in cryo-electron tomography. J Struct Biol 157:126–137PubMedCrossRefGoogle Scholar
  15. 15.
    Winkler H, Zhu P, Liu J, Ye F, Roux KH, Taylor KA (2009) Tomographic subvolume alignment and subvolume classification applied to myosin V and SIV envelope spikes. J Struct Biol 165:64–77PubMedCrossRefGoogle Scholar
  16. 16.
    Frank J (ed) (2006) Electron tomography—methods for three-dimensional visualization of structures in the cell, 2nd edn. Springer, BerlinGoogle Scholar
  17. 17.
    McIntosh JR (ed) (2007) Cellular electron microscopy, vol 79. Academic, San Diego, CAGoogle Scholar
  18. 18.
    Winkler H, Taylor KA (2003) Focus gradient correction applied to tilt series image data used in electron tomography. J Struct Biol 143:24–32PubMedCrossRefGoogle Scholar
  19. 19.
    Fernandez JJ, Li S, Crowther RA (2006) CTF determination and correction in electron cryotomography. Ultramicroscopy 106:587–596PubMedCrossRefGoogle Scholar
  20. 20.
    Hegerl R, Hoppe W (1976) Influence of electron noise on three-dimensional image reconstruction. Z Naturforsch 31a:1717–1721Google Scholar
  21. 21.
    Hoppe W, Hegerl R (1981) Some remarks concerning the influence of electron noise on 3D reconstruction. Ultramicroscopy 6:205–206Google Scholar
  22. 22.
    McEwen BF, Downing KH, Glaeser RM (1995) The relevance of dose-fractionation in tomography of radiation-sensitive specimens. Ultramicroscopy 60:357–373PubMedCrossRefGoogle Scholar
  23. 23.
    Crowther RA, Amos LA, Finch JT, De Rosier DJ, Klug A (1970) Three dimensional reconstructions of spherical viruses by Fourier synthesis from electron micrographs. Nature 226:421–425PubMedCrossRefGoogle Scholar
  24. 24.
    Saxton WO, Baumeister W, Hahn M (1984) Three-dimensional reconstruction of imperfect two-dimensional crystals. Ultramicroscopy 13:57–70PubMedCrossRefGoogle Scholar
  25. 25.
    Nickell S, Hegerl R, Baumeister W, Rachel R (2003) Pyrodictium cannulae enter the periplasmic space but do not enter the cytoplasm, as revealed by cryo-electron tomography. J Struct Biol 141:34–42PubMedCrossRefGoogle Scholar
  26. 26.
    Beck M, Forster F, Ecke M, Plitzko JM, Melchior F, Gerisch G et al (2004) Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 306:1387–1390PubMedCrossRefGoogle Scholar
  27. 27.
    Förster F, Medalia O, Zauberman N, Baumeister W, Fass D (2005) Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography. Proc Natl Acad Sci USA 102:4729–4734PubMedCrossRefGoogle Scholar
  28. 28.
    Iancu CV, Wright ER, Benjamin J, Tivol WF, Dias DP, Murphy GE et al (2005) A “flip-flop” rotation stage for routine dual-axis electron cryotomography. J Struct Biol 151:288–297PubMedCrossRefGoogle Scholar
  29. 29.
    Cheng Y, Boll W, Kirchhausen T, Harrison SC, Walz T (2007) Cryo-electron tomography of clathrin-coated vesicles: structural implications for coat assembly. J Mol Biol 365:892–899PubMedCrossRefGoogle Scholar
  30. 30.
    Penczek P, Marko M, Buttle K, Frank J (1995) Double-tilt electron tomography. Ultramicroscopy 60:393–410PubMedCrossRefGoogle Scholar
  31. 31.
    Mastronarde DN (1997) Dual-axis tomography: an approach with alignment methods that preserve resolution. J Struct Biol 120:343–352PubMedCrossRefGoogle Scholar
  32. 32.
    Chen LF, Blanc E, Chapman MS, Taylor KA (2001) Real space refinement of acto-myosin structures from sectioned muscle. J Struct Biol 133:221–232PubMedCrossRefGoogle Scholar
  33. 33.
    Lanzavecchia S, Cantele F, Bellon PL (2001) Alignment of 3D structures of macromolecular assemblies. Bioinformatics 17:58–62PubMedCrossRefGoogle Scholar
  34. 34.
    Taylor KA, Glaeser RM (1976) Electron microscopy of frozen hydrated biological specimens. J Ultrastruct Res 55:448–456PubMedCrossRefGoogle Scholar
  35. 35.
    Taylor KA, Glaeser RM (1973) Hydrophilic support films of controlled thickness and composition. Rev Sci Instrum 44:1546–1547PubMedCrossRefGoogle Scholar
  36. 36.
    Glaeser RM, Taylor KA (1978) Radiation damage relative to transmission electron microscopy of biological specimens at low temperature: a review. J Microsc 112:127–138PubMedCrossRefGoogle Scholar
  37. 37.
    Glaeser RM (1971) Limitations to significant information in biological electron microscopy as a result of radiation damage. J Ultrastruct Res 36:466–482PubMedCrossRefGoogle Scholar
  38. 38.
    Luther PK (2006) Sample shrinkage and radiation damage of plastic sections. In: Frank J (ed) Electron tomography—methods for three-dimensional visualization of structures in the cell, 2nd edn. Springer, Berlin, pp 17–48Google Scholar
  39. 39.
    Braunfeld MB, Koster AJ, Sedat JW, Agard DA (1994) Cryo automated electron tomography: towards high-resolution reconstructions of plastic-embedded structures. J Microsc 174:75–84PubMedCrossRefGoogle Scholar
  40. 40.
    Wu S, Liu J, Reedy MC, Winkler H, Reedy MK, Taylor KA (2009) Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle. J Struct Biol 168:485–502PubMedCrossRefGoogle Scholar
  41. 41.
    Taylor KA, Schmitz H, Reedy MC, Goldman YE, Franzini-Armstrong C, Sasaki H et al (1999) Tomographic 3-D reconstruction of quick frozen, Ca2+-activated contracting insect flight muscle. Cell 99:421–431PubMedCrossRefGoogle Scholar
  42. 42.
    Förster F, Pruggnaller S, Seybert A, Frangakis AS (2008) Classification of cryo-electron sub-tomograms using constrained correlation. J Struct Biol 161:276–286PubMedCrossRefGoogle Scholar
  43. 43.
    Dierksen K, Typke D, Hegerl R, Walz J, Sackmann E, Baumeister W (1995) Three-dimensional structure of lipid vesicles embedded in vitreous ice and investigated by automated electron tomography. Biophys J 68:1416–1422PubMedCrossRefGoogle Scholar
  44. 44.
    Walz J, Typke D, Nitsch M, Koster AJ, Hegerl R, Baumeister W (1997) Electron tomography of single ice-embedded macromolecules: three-dimensional alignment and classification. J Struct Biol 120:387–395PubMedCrossRefGoogle Scholar
  45. 45.
    Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76PubMedCrossRefGoogle Scholar
  46. 46.
    Brandt SS (2006) Markerless alignment in electron tomography. In: Frank J (ed) Electron tomography—methods for three-dimensional visualization of structures in the cell, 2nd edn. Springer, Berlin, pp 187–216Google Scholar
  47. 47.
    Liu J, Wright ER, Winkler H (2010) 3D visualization of HIV virions by cryoelectron tomography. Methods Enzymol 483:267–290PubMedCrossRefGoogle Scholar
  48. 48.
    Hohn M, Tang G, Goodyear G, Baldwin PR, Huang Z, Penczek PA et al (2007) SPARX, a new environment for Cryo-EM image processing. J Struct Biol 157:47–55PubMedCrossRefGoogle Scholar
  49. 49.
    Amat F, Castano-Diez D, Lawrence A, Moussavi F, Winkler H, Horowitz M (2010) Alignment of cryo-electron tomography datasets. Methods Enzymol 482:343–367PubMedCrossRefGoogle Scholar
  50. 50.
    Van Heel M, Schatz M, Orlova E (1992) Correlation functions revisited. Ultramicroscopy 46:307–316CrossRefGoogle Scholar
  51. 51.
    Horner JL, Gianino PD (1984) Phase only matched filtering. Appl Opt 23:812–816PubMedCrossRefGoogle Scholar
  52. 52.
    Saxton WO (1994) Accurate alignment of sets of images. J Microsc 174:61–68CrossRefGoogle Scholar
  53. 53.
    Vonck J (2000) Parameters affecting specimen flatness of two-dimensional crystals for electron crystallography. Ultramicroscopy 85:123–129PubMedCrossRefGoogle Scholar
  54. 54.
    Han BG, Wolf SG, Vonck J, Glaeser RM (1994) Specimen flatness of glucose-embedded biological materials for electron crystallography is affected significantly by the choice of carbon evaporation stock. Ultramicroscopy 55:1–5PubMedCrossRefGoogle Scholar
  55. 55.
    Glaeser RM (1992) Specimen flatness of thin crystalline arrays: influence of the substrate. Ultramicroscopy 46:33–43PubMedCrossRefGoogle Scholar
  56. 56.
    Kubalek EW, Kornberg RD, Darst SA (1991) Improved transfer of two-dimensional crystals from the air/water interface to specimen support grids for high-resolution analysis by electron microscopy. Ultramicroscopy 35:295–304PubMedCrossRefGoogle Scholar
  57. 57.
    Taylor DW, Kelly DF, Cheng A, Taylor KA (2007) On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy. J Struct Biol 160:305–312PubMedCrossRefGoogle Scholar
  58. 58.
    Winkler H, Taylor KA (1996) Three-dimensional distortion correction applied to tomographic reconstructions of sectioned crystals. Ultramicroscopy 63:125–132PubMedCrossRefGoogle Scholar
  59. 59.
    Crowther RA, Henderson R, Smith JM (1996) MRC image processing programs. J Struct Biol 116:9–16PubMedCrossRefGoogle Scholar
  60. 60.
    Gipson B, Zeng X, Zhang ZY, Stahlberg H (2007) 2dx—user-friendly image processing for 2D crystals. J Struct Biol 157:64–72PubMedCrossRefGoogle Scholar
  61. 61.
    Henderson R, Baldwin JM, Downing KH, Lepault J, Zemlin F (1986) Structure of purple membrane from halobacterium halobium: recording, measurement and evaluation of electron micrographs at 3.5 Å resolution. Ultramicroscopy 19:147–178CrossRefGoogle Scholar
  62. 62.
    Baldwin JM, Henderson R, Beckman E, Zemlin F (1988) Images of purple membrane at 2.8 Å resolution obtained by cryo-electron microscopy. J Mol Biol 202:585–591PubMedCrossRefGoogle Scholar
  63. 63.
    Schmid M, Dargahi R, Tam M (1993) SPECTRA: a system for processing electron images of crystals. Ultramicroscopy 48:251–264PubMedCrossRefGoogle Scholar
  64. 64.
    Frangakis AS, Bohm J, Forster F, Nickell S, Nicastro D, Typke D et al (2002) Identification of macromolecular complexes in cryoelectron tomograms of phantom cells. Proc Natl Acad Sci USA 99:14153–14158PubMedCrossRefGoogle Scholar
  65. 65.
    Ludtke SJ, Baldwin PR, Chiu W (1999) EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128:82–97PubMedCrossRefGoogle Scholar
  66. 66.
    Winkler H, Taylor KA (1999) Multivariate statistical analysis of three-dimensional cross-bridge motifs in insect flight muscle. Ultramicroscopy 77:141–152CrossRefGoogle Scholar
  67. 67.
    Bartesaghi A, Sprechmann P, Liu J, Randall G, Sapiro G, Subramaniam S (2008) Classification and 3D averaging with missing wedge correction in biological electron tomography. J Struct Biol 162:436–450PubMedCrossRefGoogle Scholar
  68. 68.
    Dube P, Tavares P, Lurz R, van Heel M (1993) The portal protein of bacteriophage SPP1: a DNA pump with 13-fold symmetry. EMBO J 12:1303–1309PubMedGoogle Scholar
  69. 69.
    Liu J, Reedy MC, Goldman YE, Franzini-Armstrong C, Sasaki H, Tregear RT et al (2004) Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges. J Struct Biol 147:268–282PubMedCrossRefGoogle Scholar
  70. 70.
    Burgess SA, Walker ML, Thirumurugan K, Trinick J, Knight PJ (2004) Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules. J Struct Biol 147:247–258PubMedCrossRefGoogle Scholar
  71. 71.
    Frank J (1990) Classification of macromolecular assemblies studied as ‘single particles’. Q Rev Biophys 23:281–329PubMedCrossRefGoogle Scholar
  72. 72.
    Pascual-Montano A, Taylor KA, Winkler H, Pascual-Marqui RD, Carazo JM (2002) Quantitative self-organizing maps for clustering electron tomograms. J Struct Biol 138:114–122PubMedCrossRefGoogle Scholar
  73. 73.
    van Heel M (1989) Classification of very large electron microscopical image data sets. Optik 82:114–126Google Scholar
  74. 74.
    Iwasaki K, Mitsuoka K, Fujiyoshi Y, Fujisawa Y, Kikuchi M, Sekiguchi K, Yamada T (2005) Electron tomography reveals diverse conformations of integrin alpha IIb beta3 in the active state. J Struct Biol 150:259–267PubMedCrossRefGoogle Scholar
  75. 75.
    Stolken M, Beck F, Haller T, Hegerl R, Gutsche I, Carazo JM et al (2011) Maximum likelihood based classification of electron tomographic data. J Struct Biol 175:77–85CrossRefGoogle Scholar
  76. 76.
    Frank J (1996) Three-dimensional elecron microscopy of macromolecular assemblies. Academic, San Diego, CAGoogle Scholar
  77. 77.
    Holmes KC, Tregear RT, Barrington Leigh J (1980) Interpretation of the low angle X-ray diffraction from insect muscle in rigor. Proc R Soc Lond B Biol Sci 207:13–33CrossRefGoogle Scholar
  78. 78.
    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612PubMedCrossRefGoogle Scholar
  79. 79.
    Jontes JD, Milligan RA (1997) Three-dimensional structure of Brush Border Myosin-I at approximately 20 resolution by electron microscopy and image analysis. J Mol Biol 266:331–342PubMedCrossRefGoogle Scholar
  80. 80.
    Wendt T, Taylor D, Trybus KM, Taylor K (2001) Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2. Proc Natl Acad Sci USA 98:4361–4366PubMedCrossRefGoogle Scholar
  81. 81.
    Liu J, Wendt T, Taylor DW, Taylor KA (2003) Refined model of the 10S conformation of smooth muscle myosin by cryoEM 3-D image reconstruction. J Mol Biol 329:963–972PubMedCrossRefGoogle Scholar
  82. 82.
    Grunewald K, Desai P, Winkler DC, Heymann JB, Belnap DM, Baumeister W, Steven AC (2003) Three-dimensional structure of herpes simplex virus from cryo-electron tomography. Science 302:1396–1398PubMedCrossRefGoogle Scholar
  83. 83.
    Zanetti G, Briggs JA, Grunewald K, Sattentau QJ, Fuller SD (2006) Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ. PLoS Pathog 2:e83PubMedCrossRefGoogle Scholar
  84. 84.
    Liu J, Bartesaghi A, Borgnia MJ, Sapiro G, Subramaniam S (2008) Molecular architecture of native HIV-1 gp120 trimers. Nature 455:109–113PubMedCrossRefGoogle Scholar
  85. 85.
    Dai W, Jia Q, Bortz E, Shah S, Liu J, Atanasov I et al (2008) Unique structures in a tumor herpes virus revealed by cryo-electron tomography and microscopy. J Struct Biol 161:428–438PubMedCrossRefGoogle Scholar
  86. 86.
    Murphy GE, Leadbetter JR, Jensen GJ (2006) In situ structure of the complete Treponema primitia flagellar motor. Nature 442:1062–1064PubMedCrossRefGoogle Scholar
  87. 87.
    Kudryashev M, Cyrklaff M, Wallich R, Baumeister W, Frischknecht F (2010) Distinct in situ structures of the Borrelia flagellar motor. J Struct Biol 169:54–61PubMedCrossRefGoogle Scholar
  88. 88.
    Nagayama K, Danev R (2009) Phase-plate electron microscopy: a novel imaging tool to reveal close-to-life nano-structures. Biophys Rev 1:37–42PubMedCrossRefGoogle Scholar
  89. 89.
    Murata K, Liu X, Danev R, Jakana J, Schmid MF, King J et al (2010) Zernike phase contrast cryo-electron microscopy and tomography for structure determination at nanometer and subnanometer resolutions. Structure 18:903–912PubMedCrossRefGoogle Scholar
  90. 90.
    Danev R, Kanamaru S, Marko M, Nagayama K (2010) Zernike phase contrast cryo-electron tomography. J Struct Biol 171:174–181PubMedCrossRefGoogle Scholar
  91. 91.
    Bouwer JC, Mackey MR, Lawrence A, Deerinck TJ, Jones YZ, Terada M et al (2004) Automated most-probable loss tomography of thick selectively stained biological specimens with quantitative measurement of resolution improvement. J Struct Biol 148:297–306PubMedCrossRefGoogle Scholar
  92. 92.
    Freitag B, Kujawa S, Mul PM, Ringnalda J, Tiemeijer PC (2005) Breaking the spherical and chromatic aberration barrier in transmission electron microscopy. Ultramicroscopy 102:209–214PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Hanspeter Winkler
    • 1
  • Shenping Wu
    • 2
  • Kenneth A. Taylor
    • 1
    Email author
  1. 1.Institute of Molecular BiophysicsFlorida State UniversityTallahasseeUSA
  2. 2.Department of Biochemistry & BiophysicsUniversity of California San FranciscoSan FranciscoUSA

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