FAUC50-β2ARH93CT4L was expressed, purified and crystallized as described in Methods. The receptor was crystallized in a cholesterol-doped (10%) monoolein cubic phase overlaid with precipitant in glass sandwich plates. Optimized precipitant consisted of 24–27 %(v/v) PEG400, 200 mM Li2SO4, 4 %(v/v) DMSO, 3.5 %(v/v) 1,4-butanediol, 100 mM MES pH 6.7. After 3–5 days of growth, crystals were harvested after adding an excess of precipitant solution for cryoprotection, and flash frozen in liquid nitrogen.
Protocol detail
Crystallization of FAUC50-β2AR(H93C)T4L
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Corresponding Organization :
Other organizations : Stanford University, University of Limerick, Friedrich-Alexander-Universität Erlangen-Nürnberg, Trinity College Dublin, D. E. Shaw Research, University of Michigan–Ann Arbor, University of Wisconsin–Madison
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Variable analysis
independent variables
- FAUC50-β2ARH93CT4L expression
- Crystallization conditions (24–27 %(v/v) PEG400, 200 mM Li2SO4, 4 %(v/v) DMSO, 3.5 %(v/v) 1,4-butanediol, 100 mM MES pH 6.7)
- Crystallization of the receptor
- Cryoprotection and flash freezing of the crystals
- Cholesterol-doped (10%) monoolein cubic phase
- Precipitant overlaid on the cubic phase
- Glass sandwich plates for crystallization
- No positive or negative controls were explicitly mentioned.
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