X-ray crystallographic analysis of the WT erα LBD-BZA complex

Running/Lysis Buffer

• 50 mM HEPES pH 8 
• 150 mM NaCl
• 5 mM BME
• 20 mM Imidazole
• 3% glycerol

Elution Buffer

• 50 mM HEPES pH 8 
• 150 mM NaCl
• 5 mM BME
• 500 mM Imidazole
• 3% glycerol

Cell Growth

• E.coli BL21 (DE3) cells with 6X-TEV-ERα ligand binding domain in pMCSG6 were grown in LB medium containing ampicillin with shaking at 37^oC until mid-log phase growth was achieved. 
• The temperature was dropped to 18oC and protein expression was induced by adding a final concentration of 400 mM IPTG. 
• Cells grew for an additional 16 hours before harvesting.
• Cells were harvest by centrifugation at 4000 xg, 15 minutes each. 

Resuspension

• Add 10 mL cold running buffer per gram cell paste.
• Use 1 EDTA-Free pic tab per 50 mL lysis.
• Thoroughly re-suspend on ice by pipetting or stirring at 4^oC.

Lysis with Sonication

• Per 50 mL lysate, 5 s on/off cycles for 5 minutes of total sonicating at 80% power on ice.

Clarifying Lysate

• Centrifuge at 18,000 xg for 30 minutes at 4^oC using the fixed angle rotor. 
• Separate and keep supernatant.

Purification

• FPLC should have the running/lysis buffer loaded on pump A and the elution buffer on pump B.
• Clean the 5 mL Bio-Rad NUVIA IMAC column by flushing 3 column volumes (CVs) of 100% pump B.
• Equilibrate the column with 5 CV of pump A or until UV baseline is reached.
• Place sample pump line into the clarified lysate and pull 5 mL through the pump to prime it and purge any air.
• Wash column with enough CVs to bring UV to baseline, elute using a linear gradient from 0-100% pump B (low to high imidazole).
• Pick peak fractions and run 20 uL (with 20 uL load dye) on a SDS-PAGE gel along with ladder.

Cleaving off the His tag using TEV Cleavage and Dialysis

• Add 1:5,000 HisTev protease, place in dialysis membrane, and dialyze against 4 L of running buffer overnight at 4^oC with stirring.

Reverse Ni-NTA (TEV Tagged)

• Place protein back over the NUVIA IMAC column and collect flow-through containing ERα ligand binding domain.

Gel Filtration

• Concentrate the protein to at least 5 mg/mL using a filtration concentrator with 30 kDa molecular weight cutoff at 4^oC.
• A final purification was performed using a Superdex 16/600 size exclusion column. 
• Peak fractions corresponding to the ER ligand binding domain dimer was pooled and concentrated to 10-15 mg/mL, flash frozen, and stored at -80^oC for later use. 

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