Recombinant human CK2α was prepared according to a reported protocol (60). The C-terminal truncated form of CK2α was cloned into the pGEX-6P-1 expression vector (GE Healthcare) and expressed in Escherichia coli strain HMS174 (DE3) (Merck) as an N-terminal glutathione S-transferase (GST)–fusion protein. GST-CK2α protein was purified by glutathione Sepharose 4B resin (GE Healthcare) and digested with PreScission Protease (80 U/ml; GE Healthcare). The GST-free CK2α protein was further purified by anion-exchange chromatography with a Mono Q column (GE Healthcare) using an AKTA purification system (GE Healthcare). The CK2α-GO289 complex was cocrystallized using 20 to 25% ethylene glycol as a precipitant, similar to our previous studies (60). Crystallographic data were collected by the MX-325HE CCD detector (Rayonix) at the beamline BL44XU of SPring-8. The dataset was processed with the program HKL2000. The initial phase was determined by the molecular replacement method using the highest-resolution CK2α structure (3WAR in the Protein Data Bank) (60) as a starting model with program MolRep. Structural modification and refinement were performed by Coot and Refmac5. Data collection and refinement statistics are shown in table S2. Maestro (Release 2017-1, Schrödinger) was used for molecular visualization.

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