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.
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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.
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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)
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Levy, C., Adams, J., Tabernero, L. (2016). Crystallization of PTP Domains. In: Pulido, R. (eds) Protein Tyrosine Phosphatases. Methods in Molecular Biology, vol 1447. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3746-2_10
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DOI: https://doi.org/10.1007/978-1-4939-3746-2_10
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