Jcsg plus screen

Screening for crystallization was performed with DYRK1A in 96-well sitting-drop plates, mixing 200 nl protein solution with 200 nl precipitant solution. Commercially available screens as well as in-house screens were used. An initial hit was found in the JCSG-plus screen (Molecular Dimensions) consisting of 0.2 M sodium thiocyanate, 20% PEG 3350. This condition was further optimized and improved by an additive screen with 96 conditions (Hampton Research), which produced the final crystallization condition: 10-16% PEG 3350, 0.1 M potassium thiocyanate, 0.1 M KCl (or 0.1 M NaCl or 0.1 M LiCl; other salts of alkali halides were also successfully used).
For co-crystallization with the inhibitor PKC412, the kinase DYRK1A was concentrated to 7-10 mg ml À1 in SEC buffer and mixed with inhibitor solution in DMSO to achieve an approximately threefold molar excess of inhibitor. The final concentration of DMSO was $4%. The protein-inhibitor mixture was then mixed in a 1:1 ratio with the crystallization solution [100 mM potassium thiocyanate, 50-100 mM LiCl (or NaCl or KCl), 10-16% PEG 3350] to give a final drop size of 4 ml. Crystallization was performed in 24-well hanging-drop plates. Octahedron-shaped crystals appeared within 5-7 d at room temperature. Crystals were cryoprotected in crystallization solution modified to include 30% ethylene glycol and were flash-cooled in liquid nitrogen.

The enzyme was crystallized at 296 K using the sitting-drop vapour-diffusion method in 96-well plates (CrystalQuick, Greiner Bio-One, Germany). Drops were prepared using 1 ml electronic reprint protein solution mixed with 1 ml reservoir solution and were equilibrated against 100 ml precipitant solution. The concentration of the protein was 10 mg ml À1 in 50 mM Tris pH 7.2. Initial crystallization condition were found using the JCSGplus screen (Molecular Dimensions) and were optimized. Diffraction-quality crystals grew within one week to approximate dimensions of 0.2 Â 0.2 Â 0.3 mm from a solution consisting of 8% PEG 8000, 8% ethylene glycol, 100 mM HEPES pH 7.5. The crystals belonged to the primitive tetragonal space group P4 1 , with unit-cell parameters a = 84.09, c = 316.38 A ˚. The asymmetric unit contained two tetramers (V M = 2.8 A ˚3 Da À1 , solvent content 56.1%) related by pseudotranslational symmetry. A native data set extending to a maximum resolution of 1.9 A ˚was collected on the BM30A beamline at ESRF, Grenoble, France using a ADSC Quantum 315r detector and a wavelength of 0.979 A ˚. For data collection a crystal of the enzyme was cooled to 100 K using a solution consisting of 15% PEG 8000, 15% ethylene glycol, 100 mM HEPES pH 7.5 as cryoprotectant. The data were processed with XDS (Kabsch, 2010).