Methods and compositions for obtaining high-resolution crystals of membrane proteins

1. A composition for lipidic cubic phase crystallization of a membrane protein, comprising

a polyethylene glycol or modified polyethylene glycol, wherein said polyethylene glycol has an average molecular weight of 300 to 2,000;

0.1 to 0.5 M of a salt;

a host lipid, wherein said host lipid is a monoacylglycerol;

a lipid additive, wherein said lipid additive is a sterol, and wherein said sterol is present at a concentration of 5-20% w/w in said host lipid;

a buffer, wherein said buffer has a pH between 4.5-8.0; and

1 to 100 mg/ml of a membrane protein.

2. The composition of claim 1 , wherein said salt is selected from the group consisting of a sulfate salt, a citrate salt, a malonate salt, a tartrate salt, an acetate salt, and a formate salt.

3. The composition of claim 2 , wherein said salt is present at a concentration of 0.1-0.2 M.

4. The composition of claim 1 , wherein said buffer is present at a concentration of 0.05-0.5 M.

5. The composition of claim 1 , further comprising an alcohol present at a concentration of 1-10% v/v.

6. The composition of claim 5 , wherein said alcohol is present at a concentration of 5-7% v/v.

7. The composition of claim 5 , wherein said alcohol is a diol or triol.

8. The composition of claim 1 wherein said host lipid is selected from the group consisting of monopalmitolein, monovaccenin and monoolein.

9. The composition of claim 1 wherein said protein to be crystallized in said composition is present at a concentration of 40-60 mg/mL.

10. The composition of claim 1 wherein said membrane protein is a G-protein coupled receptor.

11. The composition of claim 1 , wherein said membrane protein comprises a β 2 AR protein.

12. The composition of claim 11 , further comprising a ligand selected from the group consisting of carazolol, timolol, alprenolol, and clenbutorol.

13. A method of generating crystals of a membrane protein comprising:

mixing a lipid additive with a host lipid to form a lipid mixture, wherein said host lipid is a monoacylglycerol, and wherein said lipid additive is a sterol, and wherein said lipid additive is 5 to 20% w/w in said host lipid; and

combining said lipid mixture with a membrane protein solution to form a protein-lipid mixture, wherein said membrane protein is present in said protein-lipid mixture at a concentration of between 1 and 100 mg/ml; and

overlaying said protein-lipid mixture with a precipitant solution, wherein said precipitant solution comprises a polyethylene glycol or modified polyethylene glycol, wherein said polyethylene glycol has an average molecular weight of 300 to 2000; 0.1 to 0.5 M of a salt; and a buffer, wherein said buffer has a pH between 4.5-8.0.

14. A method of screening a crystal of a GPCR present in a liquid cubic phase composition comprising:

preparing a lipidic cubic phase composition, wherein said composition comprises a G-protein coupled receptor (GPCR) protein, a polyethylene glycol or modified polyethylene glycol, wherein said polyethylene glycol has an average molecular weight of 300 to 2,000; 0.1 to 0.5 M of a salt; a host lipid, wherein said host lipid is a monoacylglycerol; a lipid additive, wherein said lipid additive is a sterol and wherein said sterol is present at a concentration of 5-20% w/w in said host lipid; and a buffer, wherein said buffer has a pH between 4.5-8.0; and

exposing said composition to a first X-ray beam and determining a change in direction or intensity of said first X-ray beam;

exposing said composition to a second beam and determining a change in direction or intensity of said second X-ray beam;

identifying an area where said GPCR crystal is present in said composition; and

exposing said identified area to at least a third X-ray beam.