Pentaerythritol propoxylate

Diffracting crystals of GCPII/inhibitor complexes were obtained using procedures described previously [37 ]. Briefly, GCPII (10 mg/mL) was mixed with a stock solution of a given inhibitor in water (typically 20 – 50 mM inhibitor stock solutions) at the 10:1 (v/v) ratio, the GCPII/inhibitor solution mixed with the same volume of the reservoir solution (33% pentaerythritol propoxylate, (Sigma), 1.5% polyethylene glycol 3350 (Sigma), and 100 mM Tris–HCl, pH 8.0), and then crystallized in the hanging-drop vapor-diffusion setup at 293 K. Monocrystals of GCPII/inhibitor complexes typically appeared within one to two weeks. Crystals were flash frozen in liquid nitrogen directly from the crystallization droplets and diffraction intensities for each complex were collected from a single crystal at 100 K using synchrotron radiation at the SER-CAT beamlines 22-ID and 22-BM at the Advanced Photon Source (Argonne, USA; 1.00 Å) or at the MX 14.2 beamline (BESSYII, Helmholtz-Zentrum Berlin, Germany; 0.9181 Å). The complete dataset for each complex was collected from a single crystal and data was processed using the HKL2000 software package [38 ] or XDSAPP [39 ]. The final statistics are shown in Table 1.

The extracellular part of human GCPII (hGCPII; amino acids 44–750) was expressed in S2 cell and purified according to procedures described previously.^{29 (link)} The final protein preparation in 20 mM Tris-HCl, 150 mM NaCl, pH 8.0, was concentrated to 9 mg/mL and stored at −80 °C until further use.
Complexes of hGCPII/4 and hGCPII/6 were prepared by mixing stock solutions of hGCPII (9 mg/mL) and a given inhibitor (20 mM in water, pH adjusted to 8.0 by the addition of NaOH) at the 9:1 ratio (v/v). Crystals were grown from 2 μL droplets made by mixing equal volumes of GCPII/inhibitor and reservoir solutions (33% pentaery-thritol propoxylate (Sigma), 1.5% polyethylene glycol 3350 (Sigma), and 100 mM Tris-HCl, pH 8.0) using the hanging-drop vapor diffusion setup at 293 K. Diffraction quality crystals were flash-frozen in liquid nitrogen directly from the crystallization droplet. The diffraction data for both complexes were collected at 100 K using synchrotron radiation at the MX 14.2 beamline (BESSYII, Helmholtz-Zentrum Berlin, Germany; 0.918 Å). The complete data set was collected from a single crystal, and data were processed with the XDSAPP package.³⁸