Crystals were cryo-protected using solutions containing 75% crystallization liquor (or inhibitor soaking solution) and 25% (v/v) glycerol and frozen in liquid nitrogen prior to data collection. All data were collected from frozen crystals at 100 K. Data were acquired as 0.1° images on PILATUS 6M detectors at Diamond Light Source, UK, using beamline I03 for native data (exposure time 0.1 s per frame, beam size 80×20 μm and 30% beam transmission), and I02 for inhibitor soaked crystals (exposure time 0.05 s per frame, beam size 90×25 μm and 40% beam transmission). Diffraction images were indexed, integrated and scaled with the automated data processing program Xia2-3dii36 (link). The native data set was collected from four crystals to 2.23 Å resolution with 58-fold redundancy. A total of 7 inhibitors were soaked, including toremifene, tamoxifen, 4-hydroxyltamoxifen, raloxifene, clomiphene, ibuprofen and benztropine, and diffraction data were collected with resolutions ranging from 3.5 to 2.3 Å.
The crystals belong to space group R32 with unit cell dimensions a = b = 114.0 Å and c = 307.0 Å approximately. The apo structure was determined by molecular replacement with MOLREP37 (link) using the GP structure of the GP-KZ52 Fab complex (PDB ID, 3CSY) as a search model. There is one GP molecule in the crystal asymmetric unit. The biological trimer is formed by a crystallographic 3-fold axis. Structure refinement used REFMAC38 (link) and models were rebuilt with COOT39 (link). The apo structure was refined to 2.23 Å resolution with an Rwork of 0.223 (Rfree, 0.251) and good stereochemistry. Close examination of the data from inhibitor soaked crystals showed that only toremifene and ibuprofen were fully bound with GP, and structures were refined to resolutions of 2.69 Å and 2.68 Å, respectively. 4-hydroxyltamoxifen was only bound with partial occupancy (data not shown). Data collection and structure refinement statistics are given in Table 1. Structural comparisons used SHP40 (link), figures were prepared with PyMOL41 .
The crystals belong to space group R32 with unit cell dimensions a = b = 114.0 Å and c = 307.0 Å approximately. The apo structure was determined by molecular replacement with MOLREP37 (link) using the GP structure of the GP-KZ52 Fab complex (PDB ID, 3CSY) as a search model. There is one GP molecule in the crystal asymmetric unit. The biological trimer is formed by a crystallographic 3-fold axis. Structure refinement used REFMAC38 (link) and models were rebuilt with COOT39 (link). The apo structure was refined to 2.23 Å resolution with an Rwork of 0.223 (Rfree, 0.251) and good stereochemistry. Close examination of the data from inhibitor soaked crystals showed that only toremifene and ibuprofen were fully bound with GP, and structures were refined to resolutions of 2.69 Å and 2.68 Å, respectively. 4-hydroxyltamoxifen was only bound with partial occupancy (data not shown). Data collection and structure refinement statistics are given in Table 1. Structural comparisons used SHP40 (link), figures were prepared with PyMOL41 .
