Skip to main content
SpringerLink
Log in

Two-Dimensional Crystallization of Membrane Proteins by Reconstitution Through Dialysis

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

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

Abstract

Studies of membrane proteins by two-dimensional (2D) crystallization and electron crystallography have provided crucial information on the structure and function of a rapidly growing number of these intricate proteins within a close-to-native lipid bilayer. Here we provide protocols for planning and executing 2D crystallization trials by detergent removal through dialysis, including the preparation of phospholipids and the dialysis setup. General factors to be considered, such as the protein preparation, solubilizing detergent, lipid for reconstitution, and buffer conditions are discussed. Several 2D crystallization conditions are highlighted that have shown great promise to grow 2D crystals within a surprisingly short amount of time. Finally, conditions for optimizing order and size of 2D crystals are outlined.

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. Engel A, Hoenger A, Hefti A, Henn C, Ford RC, Kistler J, Zulauf M (1992) Assembly of 2-D membrane-protein crystals—dynamics, crystal order, and fidelity of structure-analysis by electron-microscopy. J Struct Biol 109:219–234

    Article  PubMed  CAS  Google Scholar 

  2. Jap BK, Zulauf M, Scheybani T, Hefti A, Baumeister W, Aebi U, Engel A (1992) 2D crystallization—from art to science. Ultra­microscopy 46:45–84

    Article  PubMed  CAS  Google Scholar 

  3. Dolder M, Engel A, Zulauf M (1996) The micelle to vesicle transition of lipids and detergents in the presence of a membrane protein: towards a rationale for 2D crystallization. FEBS Lett 382:203–208

    Article  PubMed  CAS  Google Scholar 

  4. Hasler L, Heymann JB, Engel A, Kistler J, Walz T (1998) 2D crystallization of membrane proteins: rationales and examples. J Struct Biol 121:162–171

    Article  PubMed  CAS  Google Scholar 

  5. Kühlbrandt W (2003) Two-dimensional crystallization of membrane proteins: a practical guide. In: Schägger H, Hunte C (eds) Membrane protein purification and crystallization: a ­practical approach. Academic Press, San Diego, pp 253–284

    Google Scholar 

  6. Schmidt-Krey I (2007) Electron crystallography of membrane proteins: two-dimensional crystallization and screening by electron microscopy. Methods 41:417–426

    Article  PubMed  CAS  Google Scholar 

  7. Abeyrathne PD, Chami M, Pantelic RS, Goldie KN, Stahlberg H (2010) Preparation of 2D crystals of membrane proteins for high-resolution electron crystallography data collection. Methods Enzymol 481:25–43

    Article  PubMed  CAS  Google Scholar 

  8. Xu C, Rice WJ, He W, Stokes DL (2002) A structural model for the catalytic cycle of Ca(2+)-ATPase. J Mol Biol 8:201–211

    Article  Google Scholar 

  9. Ryan C, Stokes DL, Chen M, Zhang Z, Hardwicke PM (2004) Effect of orthophosphate, nucleotide analogues, ADP, and phosphorylation on the cytoplasmic domains of Ca(2+)-ATPase from scallop sarcoplasmic reticulum. J Biol Chem 13:5380–5386

    Google Scholar 

  10. Stokes DL, Delavoie F, Rice WJ, Champeil P, McIntosh DB, Lacapère JJ (2005) Structural studies of a stabilized phosphoenzyme intermediate of Ca2+-ATPase. J Biol Chem 280:18063–18072

    Article  PubMed  CAS  Google Scholar 

  11. Chen Z, Akin BL, Stokes DL, Jones LR (2006) Cross-linking of C-terminal residues of phospholamban to the Ca2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain. J Biol Chem 281:14163–14172

    Article  PubMed  CAS  Google Scholar 

  12. Glaves JP, Trieber CA, Ceholski DK, Stokes DL, Young HS (2011) Phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump. J Mol Biol 405:707–723

    Article  PubMed  CAS  Google Scholar 

  13. Rigaud JL, Chami M, Lambert O, Levy D, Ranck JL (2000) Use of detergents in two-dimensional crystallization of membrane proteins. Biochim Biophys Acta Biomembr 1508:112–128

    Article  CAS  Google Scholar 

  14. Kühlbrandt W (1992) 2-dimensional crystallization of membrane proteins. Q Rev Biophys 25:1–49

    Article  PubMed  Google Scholar 

  15. Zhao G, Johnson MC, Schnell JR, Kanaoka Y, Irikura D, Lam BK, Austen KF, Schmidt-Krey I (2010) Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring a particularly large combination of specific parameters. J Struct Biol 169:450–454

    Article  PubMed  CAS  Google Scholar 

  16. Brisson A, Unwin PNT (1984) Tubular crystals of acetylcholine-receptor. J Cell Biol 99:1202–1211

    Article  PubMed  CAS  Google Scholar 

  17. Iacovache I, Biasini M, Kowal J, Kukulski W, Chami M, van der Goot FG, Engel A, Rémigy HW (2009) The 2DX robot: a membrane protein 2D crystallization Swiss Army knife. J Struct Biol 169:370–378

    Article  PubMed  Google Scholar 

  18. Kim C, Vink M, Hu M, Love J, Stokes DL, Ubarretxena-Belandia I (2010) An automated pipeline to screen membrane protein 2D crystallization. J Struct Funct Genomics 11:155–166

    Article  PubMed  CAS  Google Scholar 

  19. Hu M, Vink M, Kim C, Derr K, Koss J, D’Amico K, Cheng A, Pulokas J, Ubarretxena-Belandia I, Stokes D (2010) Automated electron microscopy for evaluating two-dimensional crystallization of membrane proteins. J Struct Biol 171(1):102–110

    Article  PubMed  CAS  Google Scholar 

  20. Rigaud JL, Mosser G, Lacapere JJ, Olofsson A, Levy D, Ranck JL (1997) Bio-beads: an efficient strategy for two-dimensional crystallization of membrane proteins. J Struct Biol 118:226–235

    Article  PubMed  CAS  Google Scholar 

  21. Schmidt-Krey I, Mutucumarana V, Haase W, Stafford DW, Kühlbrandt W (2006) Two-dimensional crystallization of human vitamin K-dependent γ-glutamyl carboxylase. J Struct Biol 157:437–442

    Article  PubMed  Google Scholar 

  22. Schmidt-Krey I, Lundqvist G, Morgenstern R, Hebert H (1998) Parameters for the two-dimensional crystallization of the membrane protein microsomal glutathione transferase. J Struct Biol 123:87–96

    Article  PubMed  CAS  Google Scholar 

  23. Schmidt-Krey I, Kanaoka Y, Mills DJ, Irikura D, Haase W, Lam BK, Austen KF, Kühlbrandt W (2004) Human leukotriene C4 synthase at 4.5 Å resolution in projection. Structure 12:2009–2014

    Article  PubMed  CAS  Google Scholar 

  24. Mitra AK, Miercke LJW, Turner GJ, Shand RF, Betlach MC, Stroud RM (1993) 2-dimensional crystallization of Escherichia-coli-expressed bacteriorhodopsin and its D96N variant—high-resolution structural studies in projection. Biophys J 65:1295–1306

    Article  PubMed  CAS  Google Scholar 

  25. Tsai CJ, Ziegler C (2005) Structure determination of secondary transport proteins by electron crystallography: two-dimensional crystallization of the betaine uptake system BetP. J Mol Microbiol Biotechnol 10:197–207

    Article  PubMed  CAS  Google Scholar 

  26. Nussberger S, Dorr K, Wang DN, Kühlbrandt W (1993) Lipid-protein interactions in crystals of plant light-harvesting complex. J Mol Biol 234:347–356

    Article  PubMed  CAS  Google Scholar 

  27. Cevc G (1987) How membrane chain melting properties are regulated by the polar surface of the lipid bilayer. Biochemistry 26:6305–6310

    Article  PubMed  CAS  Google Scholar 

  28. Mosser G (2001) Two-dimensional crystallogenesis of transmembrane proteins. Micron 32:517–540

    Article  PubMed  CAS  Google Scholar 

  29. Kruse O, Hankamer B, Konczak C, Gerle C, Morris E, Radunz A, Schmid GH, Barber J (2000) Phosphatidylglycerol is involved in the dimerization of photosystem II. J Biol Chem 275:6509–6514

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We wish to thank our colleagues in the laboratory as well as in the cryo-EM community for helpful discussions over the years. We are particularly indebted to our collaborators for entrusting us with their valuable samples. Research in the Schmidt-Krey laboratory contributing to some of these observations was funded by NIH grant HL090630 and the Integrative Biosystems Institute.

Author information

Authors and Affiliations

  1. School of Biology, Georgia Institute of Technology, Atlanta, GA, USA

    Matthew C. Johnson, Laura Y. Kim & Frederik Rudolph

  2. School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA

    Tina M. Dreaden & Bridgette A. Barry

  3. School of Biology, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA

    Ingeborg Schmidt-Krey

Authors
  1. Matthew C. Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tina M. Dreaden
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Laura Y. Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Frederik Rudolph
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bridgette A. Barry
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ingeborg Schmidt-Krey
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ingeborg Schmidt-Krey .

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

Johnson, M.C., Dreaden, T.M., Kim, L.Y., Rudolph, F., Barry, B.A., Schmidt-Krey, I. (2013). Two-Dimensional Crystallization of Membrane Proteins by Reconstitution Through Dialysis. 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_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-176-9_3

  • 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