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In situ proteolysis for protein crystallization and structure determination


We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

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  1. Huber, R., Deisenhofer, J., Colman, P.M., Matsushima, M. & Palm, W. Nature 264, 415–420 (1976).

    Article  CAS  Google Scholar 

  2. Jurnak, F., McPherson, A., Wang, A.H. & Rich, A. J. Biol. Chem. 255, 6751–6757 (1980).

    CAS  PubMed  Google Scholar 

  3. Bochkarev, A. et al. Cell 83, 39–46 (1995).

    Article  CAS  Google Scholar 

  4. Cohen, S.L., Ferre-D'Amare, A.R., Burley, S.K. & Chait, B.T. Protein Sci. 4, 1088–1099 (1995).

    Article  CAS  Google Scholar 

  5. Dong, A. et al. Nucleic Acids Res. 29, 439–448 (2001).

    Article  CAS  Google Scholar 

  6. Koth, C.M., Orlicky, S.M., Larson, S.M. & Edwards, A.M. Methods Enzymol. 368, 77–84 (2003).

    Article  CAS  Google Scholar 

  7. Campbell, E.A. et al. Mol. Cell 9, 527–539 (2002).

    Article  CAS  Google Scholar 

  8. Sawaya, M.R., Pelletier, H., Kumar, A., Wilson, S.H. & Kraut, J. Science 264, 1930–1935 (1994).

    Article  CAS  Google Scholar 

  9. Bai, Y., Auperin, T.C. & Tong, L. Acta Crystallogr. 63, 135–138 (2007).

    Article  CAS  Google Scholar 

  10. Gaur, R.K., Kupper, M.B., Fischer, R. & Hoffmann, K.M. Acta Crystallogr. 60, 965–967 (2004).

    Google Scholar 

  11. Johnson, S. et al. Acta Crystallogr. 62, 865–868 (2006).

    CAS  Google Scholar 

  12. Mandel, C.R., Gebauer, D., Zhang, H. & Tong, L. Acta Crystallogr. 62, 1041–1045 (2006).

    CAS  Google Scholar 

  13. Taneja, B., Patel, A., Slesarev, A. & Mondragon, A. EMBO J. 25, 398–408 (2006).

    Article  CAS  Google Scholar 

  14. Zhang, R.G. et al. Structure 9, 1095–1106 (2001).

    Article  CAS  Google Scholar 

  15. Xiang, S., Usunow, G., Lange, G., Busch, M. & Tong, L. J. Biol. Chem. 282, 2676–2682 (2007).

    Article  CAS  Google Scholar 

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This work was supported by US National Institutes of Health grant GM074942, by the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357, and by the Structural Genomics Consortium, which is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.

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Correspondence to Aled M Edwards.

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Dong, A., Xu, X., Edwards, A. et al. In situ proteolysis for protein crystallization and structure determination. Nat Methods 4, 1019–1021 (2007).

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