Abstract
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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References
Huber, R., Deisenhofer, J., Colman, P.M., Matsushima, M. & Palm, W. Nature 264, 415–420 (1976).
Jurnak, F., McPherson, A., Wang, A.H. & Rich, A. J. Biol. Chem. 255, 6751–6757 (1980).
Bochkarev, A. et al. Cell 83, 39–46 (1995).
Cohen, S.L., Ferre-D'Amare, A.R., Burley, S.K. & Chait, B.T. Protein Sci. 4, 1088–1099 (1995).
Dong, A. et al. Nucleic Acids Res. 29, 439–448 (2001).
Koth, C.M., Orlicky, S.M., Larson, S.M. & Edwards, A.M. Methods Enzymol. 368, 77–84 (2003).
Campbell, E.A. et al. Mol. Cell 9, 527–539 (2002).
Sawaya, M.R., Pelletier, H., Kumar, A., Wilson, S.H. & Kraut, J. Science 264, 1930–1935 (1994).
Bai, Y., Auperin, T.C. & Tong, L. Acta Crystallogr. 63, 135–138 (2007).
Gaur, R.K., Kupper, M.B., Fischer, R. & Hoffmann, K.M. Acta Crystallogr. 60, 965–967 (2004).
Johnson, S. et al. Acta Crystallogr. 62, 865–868 (2006).
Mandel, C.R., Gebauer, D., Zhang, H. & Tong, L. Acta Crystallogr. 62, 1041–1045 (2006).
Taneja, B., Patel, A., Slesarev, A. & Mondragon, A. EMBO J. 25, 398–408 (2006).
Zhang, R.G. et al. Structure 9, 1095–1106 (2001).
Xiang, S., Usunow, G., Lange, G., Busch, M. & Tong, L. J. Biol. Chem. 282, 2676–2682 (2007).
Acknowledgements
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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Dong, A., Xu, X., Edwards, A. et al. In situ proteolysis for protein crystallization and structure determination. Nat Methods 4, 1019–1021 (2007). https://doi.org/10.1038/nmeth1118
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DOI: https://doi.org/10.1038/nmeth1118
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