(sodium acetate pH 4.6, 200?mammonium acetate, 30%(= 111.1, = 38.6, = 63.3??, ?=?99.3. identity to the YefM family of antitoxins (Anantha-raman & Aravind, 2003 ?; Kamada & Hanaoka, 2005 ?). The N–terminal domain of Phd is a DNA-binding domain that is essential for autoregulation, although efficient repression also requires the presence of Doc (Magnuson from a construct (Magnuson & Yarmolinsky, 1998 ?) by PCR, while at the same time introducing gene was inserted into a pET21b vector (Novagen), which places a six-His tag at the C-terminus of BL21 (DE3) cells were subsequently transformed with pET21b-docH66Y. Cell cultures were grown in LB medium at 310?K until the OD at 600?nm was between 0.6 and 0.8. Expression of the gene was then induced by adding 1?misopropyl -d-1-thiogalactopyranoside (IPTG). 2?h after induction, the cells were harvested by centrifugation and subsequently resuspended in 20?mTrisCHCl pH 8.0, 1?mEDTA, 0.1?mg?ml?1 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF) and 1?mg?ml?1 leupeptin. Cells were broken INPP5K antibody at 277?K by passage through a cell cracker and cell debris was removed by centrifugation. The protein was loaded onto a NiCNTA affinity column equilibrated in 20?mTrisCHCl pH 8.0. The bound protein was eluted with a ten column-volume linear gradient of imidazole (0C1?NaCl. Fractions containing DocH66Y were collected and further purified on a Superdex 75 HR gel-filtration column (Amersham CGS19755 Biosciences) previously equilibrated with 20?mTrisCHCl pH 8.0. The purity of the sample was analyzed by running a 10% SDSCPAGE gel and the identity of the protein was confirmed by N-terminal sequencing (the first ten residues of the protein were sequenced and the obtained sequence was a perfect match with that expected for Doc) and Western blotting using antibodies raised in rabbits against the PhdCDoc complex (Fig. 1 ?). Open in a separate window Figure 1 Purification of DocH66Y. (TrisCHCl pH 8.0 and concentrated to 10?mg?ml?1. Concentrations were estimated spectrophotometrically at 280?nm using a theoretical molar extinction coefficient of 7450?TrisCHCl pH 7.5 (a 1:1 stoichiometry was assumed, corresponding to a calculated molar extinction coefficient of 7450?TrisCHCl pH 7.4 (a 2:1 Phd:Doc stoichiometry was assumed, corresponding to a theoretical molar extinction coefficient of 8940?TrisCHCl pH 8.0, 20% PEG 10?000 (10C108-fold dilution, with the optimal dilution being 106C107-fold). 0.3?l of these dilutions was used as an additive in the crystallization setups (which consisted of 2?l protein solution and 2?l precipitant solution). The protein concentration was lowered to 5?mg?ml?1, a concentration at which spontaneous nucleation was not observed within several weeks. 2.4. Data collection A search for a suitable cryoprotectant solution for the DocH66Y crystals was not successful. Crystals of DocH66Y were therefore mounted in thin-walled glass capillaries and X-ray data were collected at room temperature on the EMBL beamline X13 of the DESY synchrotron (Hamburg, Germany) using a 165?mm CGS19755 MAR CCD detector. Data for the DocH66YCPhd52-73Se complex were collected on EMBL beamline X12 of the DESY synchrotron using a 225?mm MAR CCD detector. The crystals CGS19755 were flash-frozen directly in the cryostream after a brief transfer (30C60?s) to a cryoprotectant solution consisting of 100?mTrisCHCl pH 8.5, 200?mNaCl, 1.5?NaBr and 35% MPD. Crystals of the PhdCDoc complex were frozen directly in the cryostream without any additional cryoprotectant. Data were initially measured to 3.2?? resolution on EMBL beamline BW7B of the DESY synchrotron using a MAR 345 image plate. Subsequently, higher resolution data were collected from the same crystal on beamline ID14-1 of the ESRF synchrotron (Grenoble, France) using an ADSC Quantum-4 detector (using two passes at 2.9 and 2.4?? resolution in order to compensate for overloads) and merged with the DESY data in order to compensate for the loss of low-resolution reflections owing to overloads. Separating reflections along axis of these crystals runs perpendicular to the plane of the plate-shaped crystals, it was not possible to orient the PhdCDoc crystals with their axis parallel to the spindle axis (which would have minimized both the spatial overlap and the rotation range necessary to obtain a complete data set). Therefore, prior to data collection the.