Computational protocol: Overexpression, crystallization and preliminary X-­ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1

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Protocol publication

[…] Crystals were grown by the sitting-drop vapour-diffusion method at 297 K by mixing equal volumes (1 µl each) of protein solution and reservoir solution. Crystals appeared after three weeks using a reservoir solution consisting of 0.1 M Tris–HCl pH 8.5, 0.2 M sodium acetate trihydrate and 30%(v/v) PEG 4000 with approximate dimensions of 0.1 × 0.1 × 0.3 mm (Fig. 2).For diffraction data collection, the crystals were directly soaked in a cryoprotectant solution composed of 30%(v/v) glycerol added to the reservoir solution. X-ray diffraction data were collected at 100 K on a Quantum 4R CCD detector (Area Detector Systems Corporation, Poway, California, USA) at the BL-6C experimental station of Pohang Light Source, Republic of Korea (Fig. 3). For each image the crystal was rotated by 1° and the raw data were processed using the HKL-2000 program suite (Otwinowski & Minor, 1997). A total of 280 647 measured reflections were merged into 40 051 unique reflections with an R merge of 12.7% and a completeness of 99.9%. The space group was determined to be P212121 on the basis of systematic absences and symmetry of diffraction intensities using the POINTLESS program from CCP4 (Winn et al., 2011). The unit-cell parameters are a = 73.96, b = 103.73, c = 114.71 Å. Table 1 summarizes the statistics of data collection. If it is assumed that eight copies of a monomer molecule are present in the crystallographic asymmetric unit, the crystal volume per protein mass (V M) is 2.38 Å3 Da−1 and the solvent content is 48.38%. Attempts to solve the structure of mANO1-CTD by the MAD method using selenomethionine-labelled mANO1-CTD or heavy-atom-derivatized crystals are in progress. […]

Pipeline specifications

Software tools HKL-2000, CCP4
Applications Small-angle scattering, Protein structure analysis
Organisms Mus musculus, Dipturus trachyderma, Escherichia coli
Chemicals Selenomethionine, Sodium