Advertisement

Crystallization of PTP Domains

  • Colin Levy
  • James Adams
  • Lydia Tabernero
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1447)

Abstract

Protein crystallography is the most powerful method to obtain atomic resolution information on the three-dimensional structure of proteins. An essential step towards determining the crystallographic structure of a protein is to produce good quality crystals from a concentrated sample of purified protein. These crystals are then used to obtain X-ray diffraction data necessary to determine the 3D structure by direct phasing or molecular replacement if the model of a homologous protein is available. Here, we describe the main approaches and techniques to obtain suitable crystals for X-ray diffraction. We include tools and guidance on how to evaluate and design the protein construct, how to prepare Se-methionine derivatized protein, how to assess the stability and quality of the sample, and how to crystallize and prepare crystals for diffraction experiments. While general strategies for protein crystallization are summarized, specific examples of the application of these strategies to the crystallization of PTP domains are discussed.

Key words

Crystallogenesis Se-methionine protein Crystal seeding Crystallization trials Cryoprotectant 

Notes

Acknowledgements

This work was supported by MRC funding (MR/K011049/1) to LT and BBSRC Doctoral Training Studentship, Sir Kenneth Murray Scholarship, Manchester Presidents Doctoral Scholar Award and a SCI Scholarship to JA. CL is a senior experimental officer at the Manchester Protein Structure Facility, University of Manchester. We thank Darel Macdonald, Andrew Currin, Efrain Ceh Pavia, and Deepankar Gahloth for sharing their data and protocols.

Supplementary material

330914_1_En_10_MOESM1_ESM.xlsx (54 kb)
Appendix: List of PTP structures in the PDB including all crystallisation conditions, expression vectors and host strains (provided as an Excel file). Crystallisation methods are listed as 1: Vapour diffusion (not stated if hanging or sitting drop); 2: Vapour diffusion hanging drop; 3: Vapour diffusion sitting drop; 4: Batch; N/A: Not stated in literature. (XLSX 53 kb)

References

  1. 1.
    Tonks NK (2006) Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol 7(11):833–846CrossRefPubMedGoogle Scholar
  2. 2.
    Tautz L, Pellecchia M, Mustelin T (2006) Targeting the PTPome in human disease. Expert Opin Ther Targets 10(1):157–177CrossRefPubMedGoogle Scholar
  3. 3.
    Eswaran J et al (2006) Crystal structures and inhibitor identification for PTPN5, PTPRR and PTPN7: a family of human MAPK-specific protein tyrosine phosphatases. Biochem J 395:483–491CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Nunes-Xavier C et al (2011) Dual-specificity MAP kinase phosphatases as targets of cancer treatment. Anticancer Agents Med Chem 11(1):109–132CrossRefPubMedGoogle Scholar
  5. 5.
    Rios P et al (2014) Dual-specificity phosphatases as molecular targets for inhibition in human disease. Antioxid Redox Signal 20(14):2251–2273CrossRefPubMedGoogle Scholar
  6. 6.
    Barr AJ et al (2009) Large-scale structural analysis of the classical human protein tyrosine phosphatome. Cell 136(2):352–363CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Tabernero L et al (2008) Protein tyrosine phosphatases: structure-function relationships. FEBS J 275(5):867–882CrossRefPubMedGoogle Scholar
  8. 8.
    Bohmer F et al (2013) Protein tyrosine phosphatase structure-function relationships in regulation and pathogenesis. FEBS J 280(2):413–431CrossRefPubMedGoogle Scholar
  9. 9.
    Berman HM et al (2000) The protein data bank. Nucleic Acids Res 28(1):235–242CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York, p A2.2Google Scholar
  11. 11.
    Mitigen. Available from http://www.mitegen.com/
  12. 12.
    Molecular Dimensions. Available from http://www.moleculardimensions.com/
  13. 13.
    Hampton Research. Available from http://hamptonresearch.com
  14. 14.
    Qiagen. Available from https://www.qiagen.com/gb/
  15. 15.
    Hampton Research, product detail. Available from http://hamptonresearch.com/product_detail.aspx?sid=152&pid=445
  16. 16.
  17. 17.
    Goldschmidt L et al (2007) Toward rational protein crystallization: a web server for the design of crystallizable protein variants. Protein Sci 16(8):1569–1576CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Cantor CR, Schimmel PR (1980) Schimmel biophysical chemistry, Part 2: Techniques for the study of biological structure and function (Pt. 2). W. H. Freeman, New YorkGoogle Scholar
  19. 19.
    Phillips K, de la Peña AH (2011) The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization. Curr Protoc Mol Biol Chapter 10:Unit 10.28Google Scholar
  20. 20.
  21. 21.
    McPherson A (2009) Introduction to macromolecular crystallography, 2nd edn. Wiley, Hoboken, NJCrossRefGoogle Scholar
  22. 22.
    Rupp B (2009) Biomolecular crystallography: principles, practice, and application to structural biology. Garland Science, New YorkGoogle Scholar
  23. 23.
    D’Arcy A, Villard F, Marsh M (2007) An automated microseed matrix-screening method for protein crystallization. Acta Crystallogr D Biol Crystallogr 63(Pt 4):550–554CrossRefPubMedGoogle Scholar
  24. 24.
  25. 25.
    Slabinski L et al (2007) XtalPred: a web server for prediction of protein crystallizability. Bioinformatics 23(24):3403–3405CrossRefPubMedGoogle Scholar
  26. 26.
    Price WN et al (2009) Understanding the physical properties that control protein crystallization by analysis of large-scale experimental data. Nat Biotechnol 27(1):51–57CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Charles M, Veesler S, Bonnete F (2006) MPCD: a new interactive on-line crystallization data bank for screening strategies. Acta Crystallogr D Biol Crystallogr 62(Pt 11):1311–1318CrossRefPubMedGoogle Scholar
  28. 28.
    Tung M, Gallagher DT (2009) The Biomolecular Crystallization Database Version 4: expanded content and new features. Acta Crystallogr D Biol Crystallogr 65:18–23CrossRefPubMedGoogle Scholar
  29. 29.
    Mesters J (2007). In: Drenth J (ed) Principles of protein X-ray crystallography. Practical Protein Cristallization. Springer, New York. pp 297–304Google Scholar
  30. 30.
    Kimber MS et al (2003) Data mining crystallization databases: knowledge-based approaches to optimize protein crystal screens. Proteins 51(4):562–568CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Manchester Protein Structure FacilityManchester Institute of BiotechnologyManchesterUK
  2. 2.Faculty of Life SciencesUniversity of ManchesterManchesterUK

Personalised recommendations