Skip to main content
SpringerLink
Log in

Image Processing of 2D Crystal Images

  • Protocol
  • First Online:
Electron Crystallography of Soluble and Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 955))

Abstract

Electron crystallography of membrane proteins uses cryo-transmission electron microscopy to image frozen-hydrated 2D crystals. The processing of recorded images exploits the periodic arrangement of the structures in the images to extract the amplitudes and phases of diffraction spots in Fourier space. However, image imperfections require a crystal unbending procedure to be applied to the image before evaluation in Fourier space. We here describe the process of 2D crystal image unbending, using the 2dx software system.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Henderson R, Unwin PN (1975) Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257:28–32

    Article  PubMed  CAS  Google Scholar 

  2. Unwin PN, Henderson R (1975) Molecular structure determination by electron microscopy of unstained crystalline specimens. J Mol Biol 94:425–440

    Article  PubMed  CAS  Google Scholar 

  3. Amos LA, Henderson R, Unwin PN (1982) Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Prog Biophys Mol Biol 39:183–231

    Article  PubMed  CAS  Google Scholar 

  4. 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 19:147–178

    Article  CAS  Google Scholar 

  5. Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E, Downing KH (1990) Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol 213:899–929

    Article  PubMed  CAS  Google Scholar 

  6. Crowther RA, Henderson R, Smith JM (1996) MRC image processing programs. J Struct Biol 116:9–16

    Article  PubMed  CAS  Google Scholar 

  7. Kühlbrandt W, Wang DN, Fujiyoshi Y (1994) Atomic model of plant light-harvesting complex by electron crystallography. Nature 367:614–621

    Article  PubMed  Google Scholar 

  8. Murata K, Mitsuoka K, Hirai T, Walz T, Agre P, Heymann JB, Engel A, Fujiyoshi Y (2000) Structural determinants of water permeation through aquaporin-1. Nature 407:599–605

    Article  PubMed  CAS  Google Scholar 

  9. Ren G, Reddy VS, Cheng A, Melnyk P, Mitra AK (2001) Visualization of a water-selective pore by electron crystallography in vitreous ice. Proc Natl Acad Sci USA 98:1398–1403

    Article  PubMed  CAS  Google Scholar 

  10. Miyazawa A, Fujiyoshi Y, Unwin N (2003) Structure and gating mechanism of the acetylcholine receptor pore. Nature 424:949–955

    Article  Google Scholar 

  11. Gonen T, Sliz P, Kistler J, Cheng Y, Walz T (2004) Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature 429:193–197

    Article  PubMed  CAS  Google Scholar 

  12. Gonen T, Cheng Y, Sliz P, Hiroaki Y, Fujiyoshi Y, Harrison SC, Walz T (2005) Lipid-protein interactions in double-layered two-dimensional AQP0 crystals. Nature 438:633–638

    Article  PubMed  CAS  Google Scholar 

  13. Hiroaki Y, Tani K, Kamegawa A, Gyobu N, Nishikawa K, Suzuki H, Walz T, Sasaki S, Mitsuoka K, Kimura K et al (2006) Implications of the aquaporin-4 structure on array formation and cell adhesion. J Mol Biol 355:628–639

    Article  PubMed  CAS  Google Scholar 

  14. Tani K, Mitsuma T, Hiroaki Y, Kamegawa A, Nishikawa K, Tanimura Y, Fujiyoshi Y (2009) Mechanism of aquaporin-4’s fast and highly selective water conduction and proton exclusion. J Mol Biol 389:694–706

    Article  PubMed  CAS  Google Scholar 

  15. Holm PJ, Bhakat P, Jegerschold C, Gyobu N, Mitsuoka K, Fujiyoshi Y, Morgenstern R, Hebert H (2006) Structural basis for detoxification and oxidative stress protection in membranes. J Mol Biol 360:934–945

    Article  PubMed  CAS  Google Scholar 

  16. Nogales E, Wolf SG, Downing KH (1998) Structure of the alpha beta tubulin dimer by electron crystallography. Nature 391:199–203

    Article  PubMed  CAS  Google Scholar 

  17. Smith JM (1999) Ximdisp – a visualization tool to aid structure determination from electron microscope images. J Struct Biol 125:223–228

    Article  PubMed  CAS  Google Scholar 

  18. Schmid MF, Dargahi R, Tam MW (1993) SPECTRA: a system for processing electron images of crystals. Ultramicroscopy 48:251–264

    Article  PubMed  CAS  Google Scholar 

  19. Hardt S, Wang B, Schmid MF (1996) A brief description of I.C.E.: the integrated crystallographic environment. J Struct Biol 116:68–70

    Article  PubMed  CAS  Google Scholar 

  20. Philippsen A, Schenk AD, Signorell GA, Mariani V, Berneche S, Engel A (2007) Collaborative EM image processing with the IPLT image processing library and toolbox. J Struct Biol 157:28–37

    Article  PubMed  CAS  Google Scholar 

  21. Philippsen A, Schenk AD, Stahlberg H, Engel A (2003) Iplt – image processing library and toolkit for the electron microscopy community. J Struct Biol 144:4–12

    Article  PubMed  Google Scholar 

  22. Sorzano CO, Marabini R, Velazquez-Muriel J, Bilbao-Castro JR, Scheres SH, Carazo JM, Pascual-Montano A (2004) XMIPP: a new generation of an open-source image processing package for electron microscopy. J Struct Biol 148:194–204

    Article  PubMed  CAS  Google Scholar 

  23. Marabini R, Masegosa IM, San Martin MC, Marco S, Fernandez JJ, de la Fraga LG, Vaquerizo C, Carazo JM (1996) Xmipp an image processing package for electron microscopy. J Struct Biol 116:237–240

    Article  PubMed  Google Scholar 

  24. Frank J, Radermacher M, Penczek P, Zhu J, Li Y, Ladjadj M, Leith A (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116:190–199

    Article  PubMed  CAS  Google Scholar 

  25. Heymann JB (2001) Bsoft: image and molecular processing in electron microscopy. J Struct Biol 133:156–169

    Article  PubMed  CAS  Google Scholar 

  26. Gipson B, Zeng X, Zhang Z, Stahlberg H (2007) 2dx – user-friendly image processing for 2D crystals. J Struct Biol 157:64–72

    Article  PubMed  CAS  Google Scholar 

  27. Gipson B, Zeng X, Stahlberg H (2007) 2dx_merge: data management and merging for 2D crystal images. J Struct Biol 160:375–384

    Article  PubMed  CAS  Google Scholar 

  28. Scherzer O (1948) The theoretical resolution limit of the electron microscope. J Appl Phys 20:20–29

    Article  Google Scholar 

  29. Smith DJ, Saxton WO, O’Keefe MA, Wood GJ, Stobbs WM (1983) The importance of beam alignment and crystal tilt in high-resolution electron microscopy. Ultramicroscopy 11:263–281

    Article  Google Scholar 

  30. Glaeser R, Downing K, DeRosier D, Chiu W, Frank J (2007) Electron crystallography of biological macromolecules. Oxford University Press, New York

    Google Scholar 

  31. Glaeser RM, Typke D, Tiemeijer PC, Pulokas J, Cheng A (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 174:1–10

    Article  PubMed  Google Scholar 

  32. Thon F (1971) Phase contrast electron microscopy. Academic, London

    Google Scholar 

  33. Philippsen A, Engel HA, Engel A (2007) The contrast-imaging function for tilted specimens. Ultramicroscopy 107:202–212

    Article  PubMed  CAS  Google Scholar 

  34. Mindell JA, Grigorieff N (2003) Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol 142:334–347

    Article  PubMed  Google Scholar 

  35. Zeng X, Gipson B, Zheng ZY, Renault L, Stahlberg H (2007) Automatic lattice determination for two-dimensional crystal images. J Struct Biol 160:353–361

    Article  PubMed  CAS  Google Scholar 

  36. Kunji ER, von Gronau S, Oesterhelt D, Henderson R (2000) The three-dimensional structure of halorhodopsin to 5 Å by electron crystallography: a new unbending procedure for two-dimensional crystals by using a global reference structure. Proc Natl Acad Sci USA 97:4637–4642

    Article  PubMed  CAS  Google Scholar 

  37. Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50:760–763

    Article  Google Scholar 

  38. Zeng X, Stahlberg H, Grigorieff N (2007) A maximum-likelihood approach to two-dimensional crystals. J Struct Biol 160:362–374

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. C-CINA, Biozentrum, University Basel, Basel, Switzerland

    Marcel Arheit, Daniel Castaño-Díez, Raphaël Thierry & Henning Stahlberg

  2. Department of Computer Sciences, Rice University, Houston, TX, USA

    Bryant R. Gipson

  3. Department of Mathematics and Computer Science, Fort Valley State University, Fort Valley, GA, USA

    Xiangyan Zeng

Authors
  1. Marcel Arheit
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Castaño-Díez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Raphaël Thierry
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bryant R. Gipson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiangyan Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Henning Stahlberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Henning Stahlberg .

Editor information

Editors and Affiliations

  1. School of Biology, Georgia Institute of Technology, Ferst Dr. 310, Atlanta, 30332, Georgia, USA

    Ingeborg Schmidt-Krey

  2. , Dept. Biochemistry & Biophysics, University of California, San Francisco, 16th St. 600, San Francisco, 94158, California, USA

    Yifan Cheng

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

Arheit, M., Castaño-Díez, D., Thierry, R., Gipson, B.R., Zeng, X., Stahlberg, H. (2013). Image Processing of 2D Crystal Images. In: Schmidt-Krey, I., Cheng, Y. (eds) Electron Crystallography of Soluble and Membrane Proteins. Methods in Molecular Biology, vol 955. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-176-9_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-176-9_10

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-175-2

  • Online ISBN: 978-1-62703-176-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation