June 28, 2010
GPCR Crystallization Conditions
With the first medically relevant GPCR structures published about 2 years ago, we can now see, for a few select examples, the 'inner workings' of GPCRs in great detail. The key to all of this was of course sample preparation, and that is: production of GPCR samples in sufficient quantity and quality and then their crystallization into well-ordered protein crystals. The fact that we can visualize GPCR molecules on an atom scale is due to identifying appropriate crystallization conditions for the respective target. Below is a compilation with the described crystallization conditions.
More to come ;)
Peter
| GPCR Target and Crystallization Technique | Protein Sample | Precipitation Reagent | TimeTemp. | PDB, Resolution, Ref. |
| human β2 adrenergicreceptor, b2AR365-Fab5 complex crystallized in DMPC/CHAPSO bicelles; hanging drop vapour diffusion | 8-12 mg/ml of b2AR-Fab5 complex; concentrated to 60 mg/ml in 10 mM HEPES pH 7.5, 100 mM NaCl, 0.1 % Dodecylmaltoside, 10 uM carazolol | Ammonium Sulphate, sodium acetate and EDTA, pH 6.5-7.5 | 7-10 days | 2R4S 3.4A REF |
| human β2 adrenergicreceptor complex with Carazolol ligand and bound Cholesterol crystallized within Lipidic Cubic Phase; receptor with T4 lysozyme fusion in 3rd intracellular loop | In Monoolein-based lipidic cubic phase, doped with ca. 8-10% Cholesterol; portions of 25-50 nL of protein-laden LCP were overlaid with 0.8uL of precipitant solution | 30-35%(v/v) PEG400, 100-200 mM NaSulfate, 100mM Bis-tris propane pH 6.5-7.0, 5-7%(v/v) 1,4-butanediol | 21-23 C | 2RH1 2.4A REF |
| human β2 adrenergicreceptor receptor with T4 lysozyme fusion in 3rd intracellular loop; Lipidic cubic phase based crystallization; receptor bound to Cholesterol and Timolol | Crystals grown in25 nL or 50 nL portions of a 10% w/w cholesterol spiked monoolein-based lipidic cubic phase. Receptor concentrated to 30 mg/mL. | 0.8 uL overlaying solution: 28% w/v PEG 400, 300 mM K Formate, 100 mM Bis-tris propane pH 7.0 and 2 mM timolol | 3D4S 2.8A REF | |
| Turkey β1 Adrenergic Receptor containing stabilizing mutations and bound Cyanopindolol crystallized by traditional vapor diffusion from micellar solution | Protein concentration 6 mg/ml | 10 mM Tris-HCL, pH 7.7; 50 mM NaCl, 0.1 mM EDTA, 0.35% Octylthiglucoside, 0.5 mM Cyanopindolol against reservoir filled with 100 mM ADA pH 6.9-7.3; 29-32% PEG600 | 2VT4 2.7A REF | |
| Human A2a Adenonsine Receptor with T4lysozyme in 3rd intracellular loop, Lipidic cubic phase based crystallization | Lipidic matrix consisting of Monoolein and Cholesterol; receptor concentrated to 70 mg/ml; In Monoolein-based lipidic cubic phase, doped with ca. 12% Cholesterol; portions of 50 nL of protein-laden LCP were overlaid with 0.8uL of precipitant solution | 30% (v/v) PEG 400 (range of 28-32%), 186 mM Lithium sulfate (range of 180 to 220 mM), 100 mM Sodium citrate (pH 6.5) (Range of 5.5 to 6.5) and 200 μM ZM241385 | 21-23 C | 3EML 2.6A Science, Science supp. |
| Methylated beta 2 Adrenergic Receptor-Fab complex | methylated receptor | 3KJ6 3.4A REF |