olecular mechanism underlying the regulation of CaMKI activity, we obtained the crystal structures of CaMKI320, CaMKI315, and CaMKI293 which diverge in the regulatory region. The structures of CaMKI320 were determined in apo form and in complex with ATP, CaMKI315 in complex with ATP, and CaMKI293 in complex with ATP, respectively. These structures are similar in the overall conformation with root mean square deviations of 0.61.6 A for 244 Ca atoms. The structure of CaMKI320-ATP is shown in Crystallization and diffraction data collection Crystallization was performed using the hanging drop vapor diffusion method. The drop consisted of equal volumes of the protein solution and the reservoir solution. The protein solution contained 10 mg/ml CaMKI protein in the lysis buffer alone or supplemented with 20 mM MgCl2 and 5 mM ATP. For the apo CaMKI320, the crystals were grown at 18uC and the reservoir solution contained 2.0 M 2SO4 and 100 mM bicine, pH 8.0. For the CaMKI320-ATP complex, the crystals were grown at 18uC and the reservoir solution contained 0.5 M 2SO4, 0.8 M Li2SO4, and 100 mM tri-sodium citrate, pH 5.6. For the CaMKI315-ATP complex, the crystals were grown at 4uC and the reservoir solution contained 1.5 M 2SO4, 3% dioxane, and 100 mM MES, pH 6.0. For the CaMKI293-ATP complex, the crystals were grown at 4uC and the reservoir solution contained 25% polyethylene glycol 8,000, 200 mM NH4Ac, and 100 mM 2AsO2Na, pH 6.0. The diffraction data of the CaMKI293-ATP complex were collected at beamline BL-6A of Photon Factory, Japan; those of the CaMKI320-ATP complex at beamline 1W2B of Beijing Synchrotron Radiation Facility, China; and those of the apo CaMKI320 and the CaMKI315-ATP complex at beamline 17U of Shanghai Synchrotron Radiation Facility, China. The data were processed, integrated, and scaled together using the MedChemExpress SB-366791 HKL2000 suite. For apo CaMKI320, the crystal used for data collection had a little bit ice, and hence during data processing the data in the resolution shell of 3.53.8 A were largely excluded which yielded a relatively low overall completeness of the whole dataset. The statistics of the diffraction data are summarized in A unusual helical conformation of the active segment Surprisingly, structural comparison shows that the apo human CaMKI320 structure obtained in this study is substantially different from the apo rat CaMKI320 structure, particularly in the activation segment, the nucleotide-binding site, and the regulatory region. In the rat CaMKI320, the activation segment is disordered and the phosphate-binding loop takes an unusual conformation with residues 2934 forming a distorted 310 helix. In addition, the regulatory region runs across the catalytic core with its C-terminus interacting with the N lobe, leading to an open conformation of the overall structure. It was proposed that the aberrant conformation of the P-loop and the obstruction of the nucleotide-binding site by helix aR2 and the aR1-aR2 loop of the regulatory region contribute to autoinhibition of the enzyme. In contrast, in our CaMKI320, the P-loop takes a canonical conformation, the Cterminus of the regulatory region is undetected, and the nucleotide-binding site is amenable to nucleotide binding. More 
intriguingly, the C-terminal part of the activation segment encompassing residues Pro171 to Thr181 assumes a unique helical conformation with well defined electron density and the DFG motif at the N-terminal part of the activation segment takes an “in”confor
