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Use of Multiple Cryoprotectants to Improve Diffraction Quality from Protein Crystals

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Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan
Japan Science and Technology Agency, CREST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
§ Division of Microbiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Biomedicinal Information Research Center and Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Koto-ku, Tokyo 135-0064, Japan
Division of Hematology and Oncology, Department of Internal Medicine, Department of Cancer Biology, Department of Neurosurgery, University of Cincinnati College of Medicine, Brain Tumor Center at University of Cincinnati Neuroscience Institute, Cincinnati, Ohio 45267, United States
# School of High Energy Accelerator Science, The Graduate University for Advanced Studies (Soken-dai), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan
*E-mail: [email protected]. Telephone: +81-29-879-6178. FAX: +81-29-879-6179.
Cite this: Cryst. Growth Des. 2016, 16, 3, 1565–1571
Publication Date (Web):January 28, 2016
Copyright © 2016 American Chemical Society

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    Abstract Image

    Current X-ray sources—such as high-flux synchrotron radiation and XFEL—enable us to determine the tertiary structures of proteins at medium or low resolution even from crystals with poor quality. However, high-resolution crystal structures are still required to obtain detailed information about protein structures in the pharmaceutical and biochemical sciences. To improve the crystal quality, several types of post-crystallization treatments, including soaking experiments, have been utilized. However, such treatments do not always improve the crystal quality, and a new method of crystal-quality enhancement is eagerly awaited. Here, we propose a novel strategy for the post-crystallization treatment, the multistep soaking method, in which a crystal is sequentially soaked in 2–3 different cryoprotectant solutions. The sequential use of different cryoprotectants significantly expands the number of options for subsequent post-crystallization treatment. Using the new method, we successfully improved the crystal quality of CagA, a type IV secretion effector derived from Helicobacter pylori, from 7.5 to 3.1 Å resolution, in addition to improving the crystal quality of phosphatidylinositol 5-phosphate 4-kinase β.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b01692.

    • Diffraction image of CagA(1–876) cryoprotected with glycerol and distribution of maximum resolutions of CagA(1–876) crystals (PDF)

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