Protein & Cell

, Volume 1, Issue 5, pp 453–458 | Cite as

Crystal structure of a novel non-Pfam protein PF2046 solved using low resolution B-factor sharpening and multi-crystal averaging methods

  • Jing Su
  • Yang Li
  • Neil Shaw
  • Weihong Zhou
  • Min Zhang
  • Hao Xu
  • Bi-Cheng Wang
  • Zhi-Jie Liu
Communication

Abstract

Sometimes crystals cannot diffract X-rays beyond 3.0 Å resolution due to the intrinsic flexibility associated with the protein. Low resolution diffraction data not only pose a challenge to structure determination, but also hamper interpretation of mechanistic details. Crystals of a 25.6 kDa non-Pfam, hypothetical protein, PF2046, diffracted X-rays to 3.38 Å resolution. A combination of Se-Met derived heavy atom positions with multiple cycles of B-factor sharpening, multi-crystal averaging, restrained refinement followed by manual inspection of electron density and model building resulted in a final model with a R value of 23.5 (Rfree= 24.7). The asymmetric unit was large and consisted of six molecules arranged as a homodimer of trimers. Analysis of the structure revealed the presence of a RNA binding domain suggesting a role for PF2046 in the processing of nucleic acids.

Keywords

low resolution diffraction PF2046 Bfactor sharpening a homodimer of trimers 

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References

  1. Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K., and Terwilliger, T.C. (2002). PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 58, 1948–1954.CrossRefGoogle Scholar
  2. Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.CrossRefGoogle Scholar
  3. Ariyoshi, M., Vassylyev, D.G., Iwasaki, H., Nakamura, H., Shinagawa, H., and Morikawa, K. (1994). Atomic structure of the RuvC resolvase: a holliday junction-specific endonuclease from E. coli. Cell 78, 1063–1072.CrossRefGoogle Scholar
  4. Bass, R.B., Strop, P., Barclay, M., and Rees, D.C. (2002). Crystal structure of Escherichia coli MscS, a voltage-modulated and mechanosensitive channel. Science 298, 1582–1587.CrossRefGoogle Scholar
  5. Bateman, A., Coin, L., Durbin, R., Finn, R.D., Hollich, V., Griffiths-Jones, S., Khanna, A., Marshall, M., Moxon, S., Sonnhammer, E. L., et al. (2004). The Pfam protein families database. Nucleic Acids Res 32, D138–141.CrossRefGoogle Scholar
  6. Berman, H.M., Bhat, T.N., Bourne, P.E., Feng, Z., Gilliland, G., Weissig, H., and Westbrook, J. (2000). The Protein Data Bank and the challenge of structural genomics. Nat Struct Biol 7 Suppl, 957–959.CrossRefGoogle Scholar
  7. Brenner, S.E. (2000). Target selection for structural genomics. Nat Struct Biol 7, 967–969.CrossRefGoogle Scholar
  8. Brenner, S.E., and Levitt, M. (2000). Expectations from structural genomics. Protein Sci 9, 197–200.CrossRefGoogle Scholar
  9. Chandonia, J.M., and Brenner, S.E. (2005). Implications of structural genomics target selection strategies: Pfam5000, whole genome, and random approaches. Proteins 58, 166–179.CrossRefGoogle Scholar
  10. Chen, B., Vogan, E.M., Gong, H., Skehel, J.J., Wiley, D.C., and Harrison, S.C. (2005). Determining the structure of an unliganded and fully glycosylated SIV gp120 envelope glycoprotein. Structure 13, 197–211.CrossRefGoogle Scholar
  11. Cowtan, K.D., and Zhang, K.Y. (1999). Density modification for macromolecular phase improvement. Prog Biophys Mol Biol 72, 245–270.CrossRefGoogle Scholar
  12. Davies, J.F. 2nd, Hostomska, Z., Hostomsky, Z., Jordan, S.R., and Matthews, D.A. (1991). Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. Science 252, 88–95.CrossRefGoogle Scholar
  13. DeLaBarre, B., and Brunger, A.T. (2003). Complete structure of p97/valosin-containing protein reveals communication between nucleotide domains. Nat Struct Biol 10, 856–863.CrossRefGoogle Scholar
  14. Dyda, F., Hickman, A.B., Jenkins, T.M., Engelman, A., Craigie, R., and Davies, D.R. (1994). Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science 266, 1981–1986.CrossRefGoogle Scholar
  15. Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126–2132.CrossRefGoogle Scholar
  16. Holm, L., Kääriäinen, S., Rosenström, P., and Schenkel, A. (2008). Searching protein structure databases with DaliLite v.3. Bioinformatics 24, 2780–2781.CrossRefGoogle Scholar
  17. Katayanagi, K., Miyagawa, M., Matsushima, M., Ishikawa, M., Kanaya, S., Ikehara, M., Matsuzaki, T., and Morikawa, K. (1990). Three-dimensional structure of ribonuclease H from E. coli. Nature 347, 306–309.CrossRefGoogle Scholar
  18. Laskowski, R.A., Watson, J.D., and Thornton, J.M. (2005). ProFunc: a server for predicting protein function from 3D structure. Nucleic Acids Res 33, W89–93.CrossRefGoogle Scholar
  19. Lopez, R., Silventoinen, V., Robinson, S., Kibria, A., and Gish, W. (2003). WU-Blast2 server at the European Bioinformatics Institute. Nucleic Acids Res 31, 3795–3798.CrossRefGoogle Scholar
  20. Ohtani, N., Yanagawa, H., Tomita, M., and Itaya, M. (2004). Cleavage of double-stranded RNA by RNase HI from a thermoacidophilic archaeon, Sulfolobus tokodaii 7. Nucleic Acids Res 32, 5809–5819.CrossRefGoogle Scholar
  21. Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276, 307–326.CrossRefGoogle Scholar
  22. Pannu, N.S., Murshudov, G.N., Dodson, E.J., and Read, R.J. (1998). Incorporation of prior phase information strengthens maximumlikelihood structure refinement. Acta Crystallogr D Biol Crystallogr 54, 1285–1294.CrossRefGoogle Scholar
  23. Pearl, F., Todd, A., Sillitoe, I., Dibley, M., Redfern, O., Lewis, T., Bennett, C., Marsden, R., Grant, A., Lee, D., et al. (2004). The CATH Domain Structure Database and related resources Gene3D and DHS provide comprehensive domain family information for genome analysis. Nucleic Acids Res 33, D247–251.CrossRefGoogle Scholar
  24. Perrakis, A., Morris, R., and Lamzin, V.S. (1999). Automated protein model building combined with iterative structure refinement. Nat Struct Biol 6, 458–463.CrossRefGoogle Scholar
  25. Rayment, I. (1997). Reductive alkylation of lysine residues to alter crystallization properties of proteins. Methods Enzymol 276, 171–179.CrossRefGoogle Scholar
  26. Rice, P., and Mizuuchi, K. (1995). Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration. Cell 82, 209–220.CrossRefGoogle Scholar
  27. Shaw, N., Cheng, C., Tempel, W., Chang, J., Ng, J., Wang, X.-Y., Perrett, S., Rose, J., Rao, Z., Wang, B.-C., et al. (2007). (NZ)CH... O contacts assist crystallization of a ParB-like nuclease. BMC Struct Biol 7, 46–58.CrossRefGoogle Scholar
  28. Walter, T.S., Meier, C., Assenberg, R., Au, K.-F., Ren, J., Verma, A., Nettleship, J.E., Owens, R.J., Stuart, D.I., and Grimes, J.M. (2006). Lysine methylation as a routine rescue strategy for protein crystallization. Structure 14, 1617–1622.CrossRefGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  • Jing Su
    • 1
  • Yang Li
    • 1
  • Neil Shaw
    • 1
  • Weihong Zhou
    • 2
  • Min Zhang
    • 3
  • Hao Xu
    • 4
  • Bi-Cheng Wang
    • 4
  • Zhi-Jie Liu
    • 1
  1. 1.National Laboratory of Biomacromolecules, Institute of BiophysicsChinese Academy of SciencesBeijingChina
  2. 2.College of Life SciencesNankai UniversityTianjinChina
  3. 3.School of Life SciencesAnhui UniversityHefeiChina
  4. 4.Department of Biochemistry and Molecular BiologyUniversity of GeorgiaAtlantaUSA

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