Laboratory scale structural genomics

  • Brent W. Segelke
  • Johana Schafer
  • Matthew A. Coleman
  • Tim P. Lekin
  • Dominique Toppani
  • Krzysztof J. Skowronek
  • Katherine A. Kantardjieff
  • Bernhard Rupp


At Lawrence Livermore National Laboratory, the development of the TB structural genomics consortium crystallization facility has paralleled several local proteomics research efforts that have grown out of gene expression microarray and comparative genomics studies. Collective experience gathered from TB consortium labs and other centers involved in the NIH-NIGMS protein structure initiative allows us to explore the possibilities and challenges of pursuing structural genomics on an academic laboratory scale. We discuss our procedures and protocols for genomic targeting approaches, primer design, cloning, small scale expression screening, scale-up and purification, through to automated crystallization screening and data collection. The procedures are carried out by a small group using a combination of traditional approaches, innovative molecular biochemistry approaches, software automation, and a modest investment in robotic equipment.

cloning crystallization expression high throughput TB structural genomics 


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  1. Alland, D., Whittam, T.S., Murray, M.B., Cave, M.D., Hazbon, M.H., Dix, K., Kokoris, M., Duesterhoeft, A., Eisen, J.A., Fraser, C.M. and Fleishman, R.D. (2003) J. Bacteriol., 185, 3392–3399.CrossRefPubMedGoogle Scholar
  2. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) J. Mol. Biol., 215, 403–410.CrossRefPubMedGoogle Scholar
  3. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Nucleic Acids Res., 25, 3389–3402.CrossRefPubMedGoogle Scholar
  4. Beernink, P.T., Segelke, B.W. and Coleman, M.A. (2003) Biochemica, 1, 4–5.Google Scholar
  5. Bertone, P., Kluger, Y., Lan, N., Zheng, D., Christendat, D., Yee, A., Edwards, A.M., Arrowsmith, C.H., Montelione, G.T. and Gerstein, M. (2001) Nucleic Acids Res., 29, 2884–2898.CrossRefPubMedGoogle Scholar
  6. Blundell, T.L., Jhoti, H. and Abell, C. (2001) Nat. Rev. Drug Discov., 1, 45–54.CrossRefGoogle Scholar
  7. Camus, J.-C., Pryor, M.J., Medigue, C. and Cole, S.T. (2002) Microbiology, 148, 2967–2973.PubMedGoogle Scholar
  8. Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V. et al. (1998) Nature, 393, 537–544.CrossRefPubMedGoogle Scholar
  9. D'Arcy, A., MacSweeney, A., Stihle, M. and Haber, A. (2003) Acta Crystallogr., D59, 396–399.Google Scholar
  10. Falquet, R.D., Pagni, M., Bucher, P., Hulo, N., Sigrist, C.J., Hofmann, K. and Bairoch, A. (2002) Nucleic Acids Res., 30, 235–238.CrossRefPubMedGoogle Scholar
  11. Fleishman, R.D., Alland, D., Eisen, J., Carpenter, L., White, O., Peterson, J., DeBoy, R., Dodson, R., Gwinn, M., Haft, D., Hickey, E., Kolonay, J.F., Nelson, W.C., Umayam, L.A., Ermolaeva, M., Salzberg, S.L., Delcher, A., Utterback, T., Weidman, J., Khouri, H., Gill, J., Mikula, A., Bishai, W., Jacobs, W.R., Jr., Venter, J.C. and Fraser, C.M. (2002) J. Bacteriol., 184, 5479–5490.CrossRefPubMedGoogle Scholar
  12. Fox, J.D., Routzahn, K.M., Bucher, M.H. and Waugh, D.S. (2003) FEBS Lett., 537, 53–57.CrossRefPubMedGoogle Scholar
  13. Goh, C.S., Lan, N., Echols, N., Douglas, S.M., Milburn, D., Bertone, P., Xiao, R., Ma, L.C., Zheng, D., Wunderlich, Z., Acton, T., Montelione, G.T. and Gerstein, M. (2003) Nucleic Acids Res., 31, 2833–2838.CrossRefPubMedGoogle Scholar
  14. Goodwill, K.E., Tennant, M.G. and Stevens, R.C. (2001) Drug Discov. Today, 6(15), S113–S118.CrossRefGoogle Scholar
  15. Goulding, C.W., Apostol, M., Anderson, D.H., Gill, S.D., Smith, C.V., Yang, J.K., Waldo, J.S., Suh, S.W., Chauhan, R., Kale, A., Bachhawat, A., Mande, S.C., Johnston, J.M., Baker, E. N., Arcus, V.L., Leys, D., McLean, K.J., Munro, A.W., Berendzen, J. and Park, M.S. (2002) Curr. Drug Targets — Infectious Disorders, 2, 121–141.CrossRefGoogle Scholar
  16. Goulding, C.W. and Perry, J.L. (2003) J. Struct. Biol., 142, 133–143.CrossRefPubMedGoogle Scholar
  17. Grossman, T.H., Kawasaki, E.S., Punreddy, S.R. and Osburne, M.S. (1998) Gene, 209, 95–103.CrossRefPubMedGoogle Scholar
  18. Haebel, P.W., Arcus, V.L., Baker, E.N. and Metcalf, P. (2001) Acta Crystallogr., D57, 1341–1343.Google Scholar
  19. Hammarstrom, M., Hellgren, N., vanDenBerg, S., Berglund, H. and Hard, T. (2002) Protein Sci., 11, 313–321.CrossRefPubMedGoogle Scholar
  20. Hansen, C.L., Skordalakes, E., Berger, J.M. and Quake, S.R. (2002) Proc. Natl. Acad. Sci. USA, 99, 16531–16536.CrossRefPubMedGoogle Scholar
  21. Harris, M. and Jones, T.A. (2002) Acta Crystallogr., D58, 1889–1891.Google Scholar
  22. Harris, T. (2001) Drug Discov. Today, 6(22), 1148.CrossRefGoogle Scholar
  23. Heinemann, U., Illing, G. and Oschkinat, H. (2001) Curr. Opin. Biotechnol., 12, 348–354.CrossRefPubMedGoogle Scholar
  24. Hennessy, D., Buchanan, B., Subramanian, D., Wilkosz, P.A. and Rosenberg, J.M. (2000) Acta Crystallogr., D56, 817–827.Google Scholar
  25. Hillier, E.S. and Lieberman, G.J. (2000) Introduction to Operations Research, McGraw-Hill, New York, NY.Google Scholar
  26. Hino, M., Shinohara, Y., Kajimoto, K., Terada, H. and Baba, Y. (2002) Protein Expr. Purif., 24, 255–259.CrossRefPubMedGoogle Scholar
  27. Hosfield, D., Palan, J., Hilgers, M., Scheibe, D., McRee, D.E. and Stevens, R.C. (2003) J. Struct. Biol., 142, 207–217.CrossRefPubMedGoogle Scholar
  28. Huang, C.C., Smith, C.V., Glickman, M.S., Jacobs, W.R., Jr. and Sacchettini, J.C. (2002) J. Biol. Chem., 277, 11559–11569.CrossRefPubMedGoogle Scholar
  29. Jancarik, J. and Kim, S.-H. (1991) J. Appl. Crystallogr., 24, 409–411.CrossRefGoogle Scholar
  30. Jones, D.T. (1999) J. Mol. Biol., 287, 797–815.CrossRefPubMedGoogle Scholar
  31. Jurisica, I., Rogers, P., Glasgow, J.I., Fortier, S., Luft, J.R., Wolfley, J.R., Bianca, M.A., Weeks, D.R. and DeTitta, G.T. (2001) IBM Systems J., 402, 248–264.Google Scholar
  32. Kantardjieff, K.A., Höchtl, P., Segelke, B.W., Tao, F.M. and Rupp, B. (2002) Acta Crystallogr., D58, 735–743.Google Scholar
  33. Kapust, R.B. and Waugh, D.S. (1999) Protein Sci., 8, 1668–1674.PubMedGoogle Scholar
  34. Kim, S.-H. (1988) Nat. Struct. Biol. Suppl, 5, 643–645.CrossRefGoogle Scholar
  35. Kimber, M.S., Vallee, F., Houston, S., Necakov, A., Skarina, T., Evdokimova, E., Beasley, S., Christendat, D., Savchenko, A., Arrowsmith, C.H., Vedadi, M., Gerstein, M. and Edwards, A.M. (2003) Proteins, 51, 562–568.CrossRefPubMedGoogle Scholar
  36. Knowles, J. and Gromo, G. (2003) Nat. Rev. Drug Targets, 2, 63–69.CrossRefGoogle Scholar
  37. Krupka, H.I., Rupp, B., Segelke, B.W., Lekin, T.P., Wright, D., Wu, H.-C., Todd, P. and Azarani, A. (2002) Acta Crystallogr., D58, 1523–1526.Google Scholar
  38. Lamzin, V.S. and Perrakis, A. (2000) Nat. Struct. Biol., 7, 979–981.CrossRefGoogle Scholar
  39. Luft, J.R., Collins, R.J., Fehrman, N.A., Lauricella, A.M., Veatch, C.K. and DeTitta, G.T. (2003) J. Struct. Biol., 142, 170–179.CrossRefPubMedGoogle Scholar
  40. Luft, J.R., Wolfley, J., Jurisica, I., Glasgow, J., Fortier, S. and DeTitta, G.T. (2001) J. Crystal Growth, 232, 591–595.CrossRefGoogle Scholar
  41. McGuffin, L.J. and Jones, D.T. (2003) Bioinformatics, 19, 874–881.PubMedGoogle Scholar
  42. McPherson, A. (1982) Preparation and Analysis of Protein Crystals, Krieger Publishing Company, Malabar, FL.Google Scholar
  43. Mulder, N.J., Apweiler, R., Attwood, T.K., Bairoch, A., Barrell, D., Bateman, A. et al. (2003) Nucleic Acids Res., 31, 315–318.CrossRefPubMedGoogle Scholar
  44. Norvell, J.C. and Zapp-Machalek, A. (2000) Nat. Struct. Biol. Suppl, 7, 931.CrossRefGoogle Scholar
  45. Page, R., Grzechnik, S.K., Canaves, J.M., Spraggon, G., Kreusch, A., Kuhn, P., Stevens, R.C. and Lesley, S.A. (2003) Acta Crystallogr., D59, 1028–1037.Google Scholar
  46. Reddy, V., Swanson, S., Segelke, B., Kantardjieff, K. A., Sacchettini, J.C. and Rupp, B. (2003) Acta Crystallogr., D59, 2200–2210.Google Scholar
  47. Rupp, B. (2003a) Acc. Chem. Res., 36, 173–181.CrossRefPubMedGoogle Scholar
  48. Rupp, B. (2003b) J. Struct. Biol., 142, 162–169.CrossRefPubMedGoogle Scholar
  49. Rupp, B., Segelke, B.W., Krupka, H.I., Lekin, T.P., Schafer, J., Zemla, A., Toppani, D., Snell, G. and Earnest, T.E. (2002) Acta Crystallogr., D58, 1514–1518.Google Scholar
  50. Santarsiero, B.D., Yegian, D.T., Lee, C.C., Spraggon, G., Gu, J., Scheibe, D., Uber, D.C., Cornell, E.W., Nordmeyer, R.A., Kolbe, W.F., Jin, J., Jones, A.L., Jaklevic, J.M., Schultz, P.G. and Stevens, R.C. (2002) J. Appl. Crystallogr., 35, 278–281.CrossRefGoogle Scholar
  51. Schroeder, E.K., de Souza, O.N., Santos, D.S., Blanchard, J.S. and Basso, L.A. (2002) Curr. Pharm. Des., 3, 197–225.CrossRefGoogle Scholar
  52. Segelke, B. and Rupp, B. (1998) ACA Meeting Series, 25, 78.Google Scholar
  53. Segelke, B.W. (2001) J. Crystal Growth, 232, 553–562.CrossRefGoogle Scholar
  54. Snell, G., Meigs, G., Cork, C., Nordmeyer, R., Cornell, E., Yegian, D., Jaklevic, J., Jin, J. and Earnest, T.E. (2002) J. Synchrotron Rad., in press.Google Scholar
  55. Spraggon, G., Lesley, S.A., Kreusch, A. and Priestle, J.P. (2002) Acta Crystallogr., D58, 1915–1923.Google Scholar
  56. Studier, F.W., Rosenberg, A.H., Dunn, J.J. and Dubendorff, J.W. (1990) Methods Enzymol., 185, 60–89.PubMedGoogle Scholar
  57. Tekaia, F., Gordon, S.V., Garnier, T., Brosch, R., Barrell, B.G. and Cole, S.T. (1999) Tubercle Lung Dis., 79, 329–342.CrossRefGoogle Scholar
  58. Terwilliger, T.C. (2000) Nat. Struct. Biol. Suppl, 7, 935–939.CrossRefGoogle Scholar
  59. Tisdall, J. (2001) Beginning Perl for Bioinformatics, O'Reilly and Associates, Sebastopol, CA.Google Scholar
  60. Waldo, G.S., Standish, B.M., Berendzen, J. and Terwilliger, T.C. (1999) Nat. Biotechnol., 17, 691–695.CrossRefPubMedGoogle Scholar
  61. Wilson, J. (2002) Acta Crystallogr., D58, 1907–1914.Google Scholar
  62. Yokoyama, S. (2003) Curr. Opin. Chem. Biol., 7, 39–43.CrossRefPubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Brent W. Segelke
    • 1
  • Johana Schafer
    • 1
  • Matthew A. Coleman
    • 2
  • Tim P. Lekin
    • 1
  • Dominique Toppani
    • 1
  • Krzysztof J. Skowronek
    • 1
  • Katherine A. Kantardjieff
    • 3
  • Bernhard Rupp
    • 1
  1. 1.Macromolecular Crystallography and Structural Genomics Group, Biology and Biotechnology Research ProgramLivermoreUSA
  2. 2.Health Effects Genetics Division, Biology and Biotechnology Research ProgramLivermoreUSA
  3. 3.W.M. Keck Foundation Center for Molecular Structure, Department of Chemistry and BiochemistryCalifornia State University FullertonFullertonUSA

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