Protein Expression and Purification. A His6-TEV-tagged ERα LBD (305-555) with a Y537S mutation in pET21a was expressed in E.coli BL21(DE3) using the previously reported methods. Cells were grown at 37 o C with shaking in LB broth, supplemented with ampicillin, until they reached mid-log phase growth (OD600 = 0.6). The temperature was reduced to 16 o C and protein expression was induced with 0.4 M IPTG for 16 h. Cells were harvested by centrifugation at 4,000 xg for 15 min then resuspended with 20 mL/g cell paste with 50 mM HEPES pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 20 mM imidazole pH 8.0, 5% glycerol, and EDTA-free PIC tabs (Pierce). Cells were lysed by sonication and cellular debris was cleared by centrifugation at 18,000 xg for 45 min. A BioRad NGC Quest FPLC was used for protein purification. The supernatant containing the His6-TEV-ERα was captured using a BioRad Nuvia IMAC column, washed with lysis buffer until baseline was reached, and eluted with 50 mM HEPES pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 500 mM imidazole pH 8.0, and 5% glycerol. 1: 5,000 mol:mol His-TEV protease was added to the protein and the solution was dialyzed against 4 L of 50 mM HEPES pH 8.0, 500 mM NaCl, 0.5 mM TCEP, pH 8.0, 5% glycerol overnight at 4 o C with stirring for 16 hours. The mixture was placed back over the IMAC column to remove the His-tag and His-TEV protease. ERα LBD Y537S was purified a final time using a GE Superdex 16/600 size exclusion column. A single peak corresponding to approximately 50 kDa MW corresponding to the ERα LBD Y537S dimer was concentrated to 10-15 mg/mL, flash frozen, and stored at -80 o C. Crystallization, X-Ray Data Collection, and Structure Solution. Each small molecule ligand was added to 10 mg/mL ERα LBD Y537S at 1 mM alongside GRIP peptide at 3 mM overnight at 4 o C. Hanging drop vapor diffusion was used to crystallize the protein using Hampton VDX plates. Clear rectangular crystals emerged after 16 h at room temperature in 15-25% PEG 3,350, 100 mM MgCl2, 100 mM Tris pH 8.0 at 5 mg/mL. Paratone-N was used as a the cryoprotectant. X-ray diffraction data were collected at the Structural Biology Center 19-BM beamline at the Advanced Photon Source, Argonne National Laboratories at 0.97 Å. Data were indexed and scaled using HKL 3,000. Phenix was used for molecular replacement using PDB 6CBZ as the input model. The structures were refined using iterative rounds of Phenix Refine and manual inspection/structure editing using Coot. Significant difference density was observed in the ligand binding pockets with one round of structure refinement that resolved after placing the compounds in the ligand binding pocket and further refinement. X-ray crystal structure collection and refinement statistics are recorded in Table . The coordinates and map were deposited in the protein databank (PDB) with accession codes 7RKE, 7SFO, and 7RMN.