Cryo-electron Microscopy of Membrane Proteins

  • Kenneth N. Goldie
  • Priyanka Abeyrathne
  • Fabian Kebbel
  • Mohamed Chami
  • Philippe Ringler
  • Henning Stahlberg
Part of the Methods in Molecular Biology book series (MIMB, volume 1117)


Electron crystallography is used to study membrane proteins in the form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.

In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.

To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beam-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.

Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base.

Key words

2D membrane protein crystals Back injection Sandwich method Spot-scanning Carbon flatness Low dose Cryo-electron microscopy 


  1. 1.
    Henderson R, Unwin PN (1975) Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257:28–32PubMedCrossRefGoogle Scholar
  2. 2.
    Unwin PN, Henderson R (1975) Molecular structure determination by electron microscopy of unstained crystalline specimens. J Mol Biol 94:425–440PubMedCrossRefGoogle Scholar
  3. 3.
    Taylor KA, Glaeser RM (1974) Electron diffraction of frozen, hydrated protein crystals. Science 186:1036–1037PubMedCrossRefGoogle Scholar
  4. 4.
    Taylor KA, Glaeser RM (1976) Electron microscopy of frozen hydrated biological specimens. J Ultrastruct Res 55:448–456PubMedCrossRefGoogle Scholar
  5. 5.
    Adrian M, Dubochet J, Lepault J et al (1984) Cryo-electron microscopy of viruses. Nature 308:32–36PubMedCrossRefGoogle Scholar
  6. 6.
    Nogales E, Wolf SG, Downing KH (1998) Structure of the alpha beta tubulin dimer by electron crystallography. Nature 391:199–203PubMedCrossRefGoogle Scholar
  7. 7.
    Henderson R, Baldwin JM, Ceska TA et al (1990) Model for the structure of Bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol 213:899–929PubMedCrossRefGoogle Scholar
  8. 8.
    Kühlbrandt W, Wang DN, Fujiyoshi Y (1994) Atomic model of plant light-harvesting complex by electron crystallography. Nature 367:614–621PubMedCrossRefGoogle Scholar
  9. 9.
    Murata K, Mitsuoka K, Hirai T et al (2000) Structural determinants of water permeation through aquaporin-1. Nature 407:599–605PubMedCrossRefGoogle Scholar
  10. 10.
    Ren G, Reddy VS, Cheng A et al (2001) Visualization of a water-selective pore by electron crystallography in vitreous ice. Proc Natl Acad Sci U S A 98:1398–1403PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Gonen T, Sliz P, Kistler J et al (2004) Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature 429:193–197PubMedCrossRefGoogle Scholar
  12. 12.
    Hiroaki Y, Tani K, Kamegawa A et al (2006) Implications of the aquaporin-4 structure on array formation and cell adhesion. J Mol Biol 355:628–639PubMedCrossRefGoogle Scholar
  13. 13.
    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 389:694–706PubMedCrossRefGoogle Scholar
  14. 14.
    Abeyrathne PD, Arheit M, Kebbel F et al (2012) Electron microscopy analysis of 2D Crystals of membrane proteins. In: Egelman EH (ed) Comprehensive biophysics. Academic, Oxford, pp 277–310CrossRefGoogle Scholar
  15. 15.
    Grigorieff N, Ceska TA, Downing KH et al (1996) Electron-crystallographic refinement of the structure of bacteriorhodopsin. J Mol Biol 259:393–421PubMedCrossRefGoogle Scholar
  16. 16.
    Mitsuoka K, Hirai T, Murata K et al (1999) The structure of bacteriorhodopsin at 3.0 Å resolution based on electron crystallography: implication of the charge distribution. J Mol Biol 286:861–882PubMedCrossRefGoogle Scholar
  17. 17.
    Dubochet J, Adrian M, Chang JJ et al (1988) Cryo-electron microscopy of vitrified specimens. Quart Rev Biophys 21:129–228CrossRefGoogle Scholar
  18. 18.
    Fujiyoshi Y, Unwin N (2008) Electron crystallography of proteins in membranes. Curr Opin Struct Biol 18:587–592PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Fujiyoshi Y, Mizusaki T, Morikawa K et al (1991) Development of a superfluid helium stage for high resolution electron microscopy. Ultramicroscopy 38:241–251CrossRefGoogle Scholar
  20. 20.
    Downing KH, Hendrickson FM (1999) Performance of a 2k CCD camera designed for electron crystallography at 400 kV. Ultramicroscopy 75:215–233PubMedCrossRefGoogle Scholar
  21. 21.
    Glaeser RM (1992) Specimen flatness of thin crystalline arrays: influence of the substrate. Ultramicroscopy 46:33–43PubMedCrossRefGoogle Scholar
  22. 22.
    Gyobu N, Tani K, Hiroaki Y et al (2004) Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique. J Struct Biol 146:325–333PubMedCrossRefGoogle Scholar
  23. 23.
    Vonck J (2000) Parameters affecting specimen flatness of two-dimensional crystals for electron crystallography. Ultramicroscopy 85:123–129PubMedCrossRefGoogle Scholar
  24. 24.
    Downing KH (1991) Spot-scan imaging in transmission electron microscopy. Science 251:53–59PubMedCrossRefGoogle Scholar
  25. 25.
    Remigy HW, Caujolle-Bert D, Suda K et al (2003) Membrane protein reconstitution and crystallization by controlled dilution. FEBS Lett 555:160–169PubMedCrossRefGoogle Scholar
  26. 26.
    Jap BK, Zulauf M, Scheybani T et al (1992) 2D crystallization: from art to science. Ultramicroscopy 46:45–84PubMedCrossRefGoogle Scholar
  27. 27.
    Levy D, Chami M, Rigaud JL (2001) Two-dimensional crystallization of membrane proteins: the lipid layer strategy. FEBS Lett 504:187–193PubMedCrossRefGoogle Scholar
  28. 28.
    Kühlbrandt W (1992) Two-dimensional crystallization of membrane proteins. Quart Rev Biophys 25:1–49CrossRefGoogle Scholar
  29. 29.
    Hasler L, Heymann JB, Engel A et al (1998) 2D crystallization of membrane proteins: rationales and examples. J Struct Biol 121:162–171PubMedCrossRefGoogle Scholar
  30. 30.
    Abeyrathne PD, Chami M, Pantelic RS et al (2010) Preparation of 2D crystals of membrane proteins for high-resolution electron crystallography data collection. Meth Enzymol 481:25–43PubMedCrossRefGoogle Scholar
  31. 31.
    Signorell GA, Kaufmann TC, Kukulski W et al (2007) Controlled 2D crystallization of membrane proteins using methyl-beta-cyclodextrin. J Struct Biol 157:321–328PubMedCrossRefGoogle Scholar
  32. 32.
    Iacovache I, Biasini M, Kowal J et al (2010) The 2DX robot: a membrane protein 2D crystallization Swiss Army knife. J Struct Biol 169:370–378PubMedCrossRefGoogle Scholar
  33. 33.
    Coudray N, Hermann G, Caujolle-Bert D et al (2011) Automated screening of 2D crystallization trials using transmission electron microscopy: a high-throughput tool-chain for sample preparation and microscopic analysis. J Struct Biol 173:365–374PubMedCrossRefGoogle Scholar
  34. 34.
    Hu M, Vink M, Kim C et al (2010) Automated electron microscopy for evaluating two-dimensional crystallization of membrane proteins. J Struct Biol 171:102–110PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Henderson R (1992) Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice. Ultramicroscopy 46:1–18PubMedCrossRefGoogle Scholar
  36. 36.
    Kimura Y, Vassylyev DG, Miyazawa A et al (1997) Surface of bacteriorhodopsin revealed by high-resolution electron crystallography. Nature 389:206–211PubMedCrossRefGoogle Scholar
  37. 37.
    Glaeser RM (2008) Retrospective: radiation damage and its associated “information limitations”. J Struct Biol 163:271–276PubMedCrossRefGoogle Scholar
  38. 38.
    Golas MM, Sander B, Will CL et al (2003) Molecular architecture of the multiprotein splicing factor SF3b. Science 300:980–984PubMedCrossRefGoogle Scholar
  39. 39.
    Golas MM, Sander B, Will CL et al (2005) Major conformational change in the complex SF3b upon integration into the spliceosomal U11/U12 di-snRNP as revealed by electron cryomicroscopy. Mol Cell 17:869–883PubMedCrossRefGoogle Scholar
  40. 40.
    Glaeser RM, Typke D, Tiemeijer PC et al (2011) Precise beam-tilt alignment and collimation are required to minimize the phase error associated with coma in high-resolution cryo-EM. J Struct Biol 174:1–10PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Aebi U, Smith PR, Dubochet J et al (1973) A study of the structure of the T-layer of Bacillus brevis. J Supramol Struct 1:498–522PubMedCrossRefGoogle Scholar
  42. 42.
    Glaeser RM, Hall RJ (2011) Reaching the information limit in cryo-EM of biological macromolecules: experimental aspects. Biophys J 100:2331–2337PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Zhang X, Zhou ZH (2011) Limiting factors in atomic resolution cryo electron microscopy: no simple tricks. J Struct Biol 175:253–263PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Walz T, Grigorieff N (1998) Electron crystallography of two-dimensional crystals of membrane proteins. J Struct Biol 121:142–161PubMedCrossRefGoogle Scholar
  45. 45.
    Downing KH, Li H (2001) Accurate recording and measurement of electron diffraction data in structural and difference Fourier studies of proteins. Microsc Microanal 7:407–417PubMedCrossRefGoogle Scholar
  46. 46.
    Iancu CV, Wright ER, Heymann JB et al (2006) A comparison of liquid nitrogen and liquid helium as cryogens for electron cryotomography. J Struct Biol 153:231–240PubMedCrossRefGoogle Scholar
  47. 47.
    Comolli LR, Downing KH (2005) Dose tolerance at helium and nitrogen temperatures for whole cell electron tomography. J Struct Biol 152:149–156PubMedCrossRefGoogle Scholar
  48. 48.
    Bammes BE, Jakana J, Schmid MF et al (2010) Radiation damage effects at four specimen temperatures from 4 to 100 K. J Struct Biol 169:331–341PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Fujiyoshi Y (1998) The structural study of membrane proteins by electron crystallography. Adv Biophys 35:25–80PubMedCrossRefGoogle Scholar
  50. 50.
    Amos LA, Henderson R, Unwin PN (1982) Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Prog Biophys Mol Biol 39:183–231PubMedCrossRefGoogle Scholar
  51. 51.
    Henderson R, Baldwin JM, Downing KH et al (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
  52. 52.
    Crowther R, Henderson R, Smith J (1996) MRC image processing programs. J Struct Biol 116:9–16PubMedCrossRefGoogle Scholar
  53. 53.
    Gipson B, Zeng X, Zhang Z et al (2007) 2dx—user-friendly image processing for 2D crystals. J Struct Biol 157:64–72PubMedCrossRefGoogle Scholar
  54. 54.
    Gipson B, Zeng X, Stahlberg H (2008) 2dx - automated 3D structure reconstruction from 2D crystal data. Microsc Microanal 14:1290–1291CrossRefGoogle Scholar
  55. 55.
    Gipson B, Zeng X, Stahlberg H (2007) 2dx_merge: data management and merging for 2D crystal images. J Struct Biol 160:375–384PubMedCentralPubMedCrossRefGoogle Scholar
  56. 56.
    Zeng X, Gipson B, Zheng ZY et al (2007) Automatic lattice determination for two-dimensional crystal images. J Struct Biol 160:353–361PubMedCentralPubMedCrossRefGoogle Scholar
  57. 57.
    Zeng X, Stahlberg H, Grigorieff N (2007) A maximum likelihood approach to two-dimensional crystals. J Struct Biol 160:362–374PubMedCentralPubMedCrossRefGoogle Scholar
  58. 58.
    Philippsen A, Schenk AD, Signorell GA et al (2007) Collaborative EM image processing with the IPLT image processing library and toolbox. J Struct Biol 157:28–37PubMedCrossRefGoogle Scholar
  59. 59.
    Philippsen A, Schenk AD, Stahlberg H et al (2003) IPLT – image processing library and toolkit for the electron microscopy community. J Struct Biol 144:4–12PubMedCrossRefGoogle Scholar
  60. 60.
    Schenk AD, Castano-Diez D, Gipson B et al (2010) 3D reconstruction from 2D crystal image and diffraction data. Meth Enzymol 482:101–129PubMedCrossRefGoogle Scholar
  61. 61.
    Schmidt-Krey I, Cheng Y (eds) (2013) Electron crystallography of soluble and membrane proteins, vol 95, Methods in Molecular Biology. Humana Press, New York, NYGoogle Scholar
  62. 62.
    Arheit M, Castano-Diez D, Thierry R et al (2013) Merging of image data in electron crystallography. Meth Mol Biol 955:195–209CrossRefGoogle Scholar
  63. 63.
    Arheit M, Castano-Diez D, Thierry R et al (2013) Automation of image processing in electron crystallography. Meth Mol Biol 955:313–330CrossRefGoogle Scholar
  64. 64.
    Arheit M, Castano-Diez D, Thierry R et al (2013) Image processing of 2D crystal images. Meth Mol Biol 955:171–194CrossRefGoogle Scholar
  65. 65.
    Glaeser RM, Downing KH (1992) Assessment of resolution in biological electron crystallography. Ultramicroscopy 47:256–265PubMedCrossRefGoogle Scholar
  66. 66.
    Glaeser RM, Downing KH (2004) Specimen charging on thin films with one conducting layer: discussion of physical principles. Microsc Microanal 10:790–796PubMedCrossRefGoogle Scholar
  67. 67.
    Butt H-J, Wang DN, Hansma PK et al (1991) Effect of surface roughness of carbon support films on high-resolution electron diffraction of two-dimensional protein crystals. Ultra-microscopy 36:307–318Google Scholar
  68. 68.
    Booy FP, Pawley JB (1993) Cryo-crinkling: what happens to carbon films on copper grids at low temperature. Ultramicroscopy 48:273–280PubMedCrossRefGoogle Scholar
  69. 69.
    Mindell JA, Maduke M, Miller C et al (2001) Projection structure of a ClC-type chloride channel at 6.5 Å resolution. Nature 409:219–223PubMedCrossRefGoogle Scholar
  70. 70.
    Stahlberg H, Braun T, de Groot B et al (2000) The 6.9 Å structure of GlpF: a basis for homology modeling of the glycerol channel from Escherichia coli. J Struct Biol 132:133–141PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Kenneth N. Goldie
    • 1
  • Priyanka Abeyrathne
    • 1
  • Fabian Kebbel
    • 1
  • Mohamed Chami
    • 1
  • Philippe Ringler
    • 1
  • Henning Stahlberg
    • 2
  1. 1.Center for Cellular Imaging and NanoAnalytics (C-CINA), BiozentrumUniversity BaselBaselSwitzerland
  2. 2.Center for Cellular Imaging and NanoAnalytics (C-CINA), BiozentrumUniversity of BaselBaselSwitzerland

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