Phasing Electron Diffraction Data by Molecular Replacement: Strategy for Structure Determination and Refinement

  • Goragot Wisedchaisri
  • Tamir GonenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 955)


Electron crystallography is arguably the only electron cryomicroscopy (cryo EM) technique able to deliver atomic resolution data (better then 3 Å) for membrane proteins embedded in a membrane. The progress in hardware improvements and sample preparation for diffraction analysis resulted in a number of recent examples where increasingly higher resolutions were achieved. Other chapters in this book detail the improvements in hardware and delve into the intricate art of sample preparation for microscopy and electron diffraction data collection and processing. In this chapter, we describe in detail the protocols for molecular replacement for electron diffraction studies. The use of a search model for phasing electron diffraction data essentially eliminates the need of acquiring image data rendering it immune to aberrations from drift and charging effects that effectively lower the attainable resolution.

Key words

Electron cryomicroscopy (Cryo-EM) Electron crystallography Electron diffraction Molecular replacement Structure refinement 



Research in the Gonen laboratory is supported by the American Diabetes Association Award # 1-09-CD-05 and by the National Institutes of Health R01GM079233 and U54GM094598 as well as the Howard Hughes Medical Institute.


  1. 1.
    Henderson R, Unwin PN (1975) Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257:28–32PubMedCrossRefGoogle Scholar
  2. 2.
    Fujiyoshi Y (1998) The structural study of membrane proteins by electron crystallography. Adv Biophys 35:25–80PubMedCrossRefGoogle Scholar
  3. 3.
    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 438:633–638PubMedCrossRefGoogle Scholar
  4. 4.
    Hite RK, Li Z, Walz T (2010) Principles of membrane protein interactions with annular lipids deduced from aquaporin-0 2D crystals. EMBO J 29:1652–1658PubMedCrossRefGoogle Scholar
  5. 5.
    Mitsuoka K, Hirai T, Murata K, Miyazawa A, Kidera A, Kimura Y, Fujiyoshi Y (1999) The structure of bacteriorhodopsin at 3.0 A resolution based on electron crystallography: implication of the charge distribution. J Mol Biol 286:861–882PubMedCrossRefGoogle Scholar
  6. 6.
    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 389:694–706PubMedCrossRefGoogle Scholar
  7. 7.
    Crowther RA, Henderson R, Smith JM (1996) MRC image processing programs. J Struct Biol 116:9–16PubMedCrossRefGoogle Scholar
  8. 8.
    Gipson B, Zeng X, Stahlberg H (2007) 2dx_merge: data management and merging for 2D crystal images. J Struct Biol 160:375–384PubMedCrossRefGoogle Scholar
  9. 9.
    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 157:28–37PubMedCrossRefGoogle Scholar
  10. 10.
    Ceska TA, Henderson R (1990) Analysis of high-resolution electron diffraction patterns from purple membrane labelled with heavy-atoms. J Mol Biol 213:539–560PubMedCrossRefGoogle Scholar
  11. 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 429:193–197PubMedCrossRefGoogle Scholar
  12. 12.
    Navaza J (1994) Amore—an automated package for molecular replacement. Acta Crystallogr A50:157–163Google Scholar
  13. 13.
    Trapani S, Navaza J (2008) AMoRe: classical and modern. Acta Crystallogr D64:11–16Google Scholar
  14. 14.
    Vagin A, Teplyakov A (1997) MOLREP: an automated program for molecular replacement. J Appl Crystallogr 30:1022–1025CrossRefGoogle Scholar
  15. 15.
    Vagin A, Teplyakov A (2010) Molecular replacement with MOLREP. Acta Crystallogr D66:22–25Google Scholar
  16. 16.
    McCoy AJ (2007) Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr D63:32–41Google Scholar
  17. 17.
    McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674PubMedCrossRefGoogle Scholar
  18. 18.
    Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D50:760–763Google Scholar
  19. 19.
    Brunger AT (2007) Version 1.2 of the crystallography and NMR system. Nat Protoc 2:2728–2733PubMedCrossRefGoogle Scholar
  20. 20.
    Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D54:905–921Google Scholar
  21. 21.
    Potterton E, Briggs P, Turkenburg M, Dodson E (2003) A graphical user interface to the CCP4 program suite. Acta Crystallogr D59:1131–1137Google Scholar
  22. 22.
    Stein N (2008) CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J Appl Crystallogr 41:641–643CrossRefGoogle Scholar
  23. 23.
    Vagin A, Teplyakov A (2000) An approach to multi-copy search in molecular replacement. Acta Crystallogr D56:1622–1624Google Scholar
  24. 24.
    Tong L (2001) How to take advantage of non-crystallographic symmetry in molecular replacement: ‘locked’ rotation and translation functions. Acta Crystallogr D57:1383–1389Google Scholar
  25. 25.
    Tong L, Rossmann MG (1990) The locked rotation function. Acta Crystallogr A46:783–792Google Scholar
  26. 26.
    Tong LA (1996) The locked translation function and other applications of a Patterson correlation function. Acta Crystallogr A52:476–479Google Scholar
  27. 27.
    Long F, Vagin AA, Young P, Murshudov GN (2008) BALBES: a molecular-replacement pipeline. Acta Crystallogr D64:125–132Google Scholar
  28. 28.
    Keegan RM, Winn MD (2008) MrBUMP: an automated pipeline for molecular replacement. Acta Crystallogr D64:119–124Google Scholar
  29. 29.
    Sui HX, Han BG, Lee JK, Walian P, Jap BK (2001) Structural basis of water-specific transport through the AQP1 water channel. Nature 414:872–878PubMedCrossRefGoogle Scholar
  30. 30.
    Vaguine AA, Richelle J, Wodak SJ (1999) SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta Crystallogr D55:191–205Google Scholar
  31. 31.
    French S, Wilson K (1978) Treatment of negative intensity observations. Acta Crystallogr A34:517–525Google Scholar
  32. 32.
    Matthews BW (1968) Solvent content of protein crystals. J Mol Biol 33:491–497PubMedCrossRefGoogle Scholar
  33. 33.
    Kantardjieff KA, Rupp B (2003) Matthews coefficient probabilities: improved estimates for unit cell contents of proteins, DNA, and protein-nucleic acid complex crystals. Protein Sci 12:1865–1871PubMedCrossRefGoogle Scholar
  34. 34.
    Teneyck LF (1973) Crystallographic fast Fourier-transforms. Acta Crystallogr A29:183–191Google Scholar
  35. 35.
    Lebedev AA, Vagin AA, Murshudov GN (2008) Model preparation in MOLREP and examples of model improvement using X-ray data. Acta Crystallogr D64:33–39Google Scholar
  36. 36.
    Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D60:2126–2132Google Scholar
  37. 37.
    Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D66:486–501Google Scholar
  38. 38.
    Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D53:240–255Google Scholar
  39. 39.
    Cohen SX, Ben Jelloul M, Long F, Vagin A, Knipscheer P, Lebbink J, Sixma TK, Lamzin VS, Murshudov GN, Perrakis A (2008) ARP/wARP and molecular replacement: the next generation. Acta Crystallogr D64:49–60Google Scholar
  40. 40.
    Perrakis A, Harkiolaki M, Wilson KS, Lamzin VS (2001) ARP/wARP and molecular replacement. Acta Crystallogr D57:1445–1450Google Scholar
  41. 41.
    Terwilliger TC (2003) Improving macromolecular atomic models at moderate resolution by automated iterative model building, statistical density modification and refinement. Acta Crystallogr D59:1174–1182Google Scholar
  42. 42.
    Terwilliger TC (2003) Automated main-chain model building by template matching and iterative fragment extension. Acta Crystallogr D59:38–44Google Scholar
  43. 43.
    Terwilliger TC (2003) Automated side-chain model building and sequence assignment by template matching. Acta Crystallogr D59:45–49Google Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Janelia Farm Research Campus, Howard Hughes Medical InstituteAshburnUSA

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