Gen) for 72 h at 27 . The protein was puri d applying a mixture of TALON (Clontech) metal af ity chromatography, ammonium Doxycycline (monohydrate) manufacturer sulfate precipitation, S200 size exclusion chromatography and ally S75 size exclusion chromatography (Amersham Biotech.). The His6 tag was removed from the protein following the ammonium sulfate precipitation by cleavage with TEV protease. The protein was isolated in the al puri ation step in 10 mM Tris Cl pH 8.0, 750 mM NaCl, five glycerol, 3 mM dithiothreitol (DTT) and 1 mM EDTA. Restricted proteolysis experiments had been performed working with trypsin, subtilisin and thermolysin. Cdc14B (two mg/ml) in a buffer of 90 mM Tris Cl pH eight.5, 750 mM NaCl, four mM DTT, two mM CaCl2 was digested for 30 min with each of 0.1, 0.5, 1, five, 10 or 20 mg/ml protease. The 40 kDa big 41bbl Inhibitors Related Products product in the digests was characterized by Nterminal sequencing, indicating an Nterminus at residue Pro44. The Cterminal boundary was estimated as His386 on the basis of molecular weight and secondary structure predictions. A pFASTBAC vector containing the coding sequence for Pro44 is386 was constructed and termed pFBCdc14B. Many mutations have been produced to pFBCdc14B applying standard sitedirected mutagenesis methods. Cdc14B and Cdc14B mutants were expressed in Sf9 cells and puri d as described for the fulllength protein, except that the ammonium sulfate precipitation step was omitted. Crystals of Cdc14B had been obtained by hanging drop vapour diffusion by mixing 1 ml of five mg/ml protein with 1 ml of properly resolution (of 700 ml) comprising one hundred mM Trisacetate pH 8.0, 30 polyethylene glycol (PEG) 4000, 0.2 M MgCl2 and 7 mM DTT incubated at 20 . These crystals had been improved by microseeding into 100 mM Trisacetate pH 8.0, 23 PEG 4000, 0.two M MgCl2, 7 mM DTT. Crystals containing a bound substrate have been developed by mixing a phosphopeptide (acetylAlapSerProArgArgArgamide) with protein at a three:1 molar ratio and crystallizing in 0.1 M Trisacetate, 30 PEG 1500 at 20 . For data collection at 100 K, crystals were transferred to a cryoprotectant buffer of 100 mM Tris Cl pH eight.0, 20 PEG 4000, ten MPD, 0.4 M NaCl, 0.21 M MgCl2 and five glycerol. At a concentration of 20 mM Na2WO4, the activity of Cdc14B working with 5 mM pNPP as a substrate is absolutely inhibited. We for that reason made use of Na2WO4 to type a Cdc14B ungstate derivative by supplementing the cryoprotectant with 25 mM Na2WO4. Structure determination The 39.7 kDa Cdc14B fragment crystallized in space group C2 with 1 molecule per asymmetric unit. The structure was determined by the SAD process using WO4labelled crystals from data collected in the ID14 beamlines, ESRF, Grenoble, France. All crystallographic data have been processed utilizing MOSFLM (CCP4, 1994) and SCALA (CCP4, 1994). WO4 heavy atom coordinates had been identi d using Resolve (Terwilliger and Berendzen, 1999) plus the parameters have been re ed and protein phase calculations were performed working with SHARP (De la Fortelle and Bricogne, 1997). The improved phases from SOLOMON (Abrahams and Leslie, 1996) have been used to calculate electron density maps that were readily interpretable. Crystallographic statistics are listed in Table I. Electron density map interpretation and model building had been performed working with the system O (Jones et al., 1991) and the structure was re ed by CNS (Brunger et al., 1998). Electron density maps for the structure of the phosphopeptide substrate bound to Cdc14B Cys314 er were generated by re ing the model with the apo enzyme against the information from the substratebound crystals utilizing CNS.
