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Report No.

Preparation of large-volume crystals for structure analysis of human casein kinase-2 by neutron crystallography

Shibazaki, Chie; Adachi, Motoyasu; Hiromoto, Takeshi; Shimizu, Rumi; Kuroki, Ryota

Casein kinase 2 (CK2) is one of the ubiquitous Ser/Thr kinases and is involved in the cell cycle and the survival and proliferation of cells. CK2 is a heterotetrameric structure comprising two $$alpha$$- or $$alpha$$-subunits and two regulatory $$beta$$-subunits. In order to understand the biological function of the alpha catalytic subunit of CK2$$alpha$$, we aim to analyze the structure of CK2$$alpha$$ including information of the hydrogen and hydrating water molecule by neutron crystallography. The gene coding CK2$$alpha$$ was inserted into pET24a and expressed in E. coli strain BL21DE3, in which the mobile region and chemically reactive thiols were removed by amino acid mutation. A total of 150 mg protein was obtained from a 6 L culture, and was used for crystallization trials. The preparation of large crystals was performed using a macro seeding method specially developed for CK2$$alpha$$. Finally, a large crystal with a volume of approximately 2 mm$$^{3}$$ was reproducibly obtained. From the X-ray diffraction study, we confirmed that the crystals obtained diffracted to approx. 1 ${AA}$ resolution at 100 K after soaking the crystal into the deuterated cryo protectant. The neutron diffraction data collection is planned to obtain a high resolution neutron structure of CK2$$alpha$$.



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