Pressure cryocooling protein crystals
AssigneeCornell Research Foundation, Inc.
InventorsChae Un Kim, Sol Gruner
Life of this patent
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Abstract
Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.
Claims5
A method for cryoprotection of a protein crystal containing water surrounding and inside the crystal allowing collection of high resolution data by X-ray crystallography analysis, comprising the steps of
- (a) coating the crystal to prevent dehydration,
- (b) pressurizing the crystal with a noble gas, to a pressure ranging from 100 to 800 MPa, at a temperature ranging from above the freezing point of the water in the crystal to room temperature, for a time period sufficient for the crystal to equilibrate,
- (c) cooling the noble gas pressurized crystal to below 120 K, and
- (d) releasing the noble gas pressure and maintaining the crystal at cryogenic temperature.
A method for cryoprotection of a protein crystal containing water surrounding and inside the crystals allowing collection of high resolution data by x-ray crystallography analysis, comprising the steps of
- (a) growing a crystal in aqueous solution, in a capillary,
- (b) pressurizing the entire capillary in which the crystal was grown in crystallization solution, to a pressure ranging from 100 to 800 MPa, for a time period sufficient for the crystal to equilibrate,
- (c) cooling the noble gas pressurized capillary containing crystal in solution to below 120 K, and
- (d) releasing the noble gas pressure, and maintaining the capillary containing crystal in solution at cryogenic temperature for x-ray analysis.
A method for producing a cryocooled protein crystal for obtaining high resolution data and for obtaining phase information by single anomalous diffraction (SAD) simultaneously, comprising the steps of
- (a) providing a protein crystal which contains water surrounding and inside it,
- (b) coating the crystal to prevent dehydration,
- (c) pressurizing the crystal at ambient temperature with a heavier noble gas which has binding sites in the crystal at a pressure sufficient to obtain binding by the noble gas to produce a sufficient anomalous signal for phasing,
- (d) releasing the pressure of the pressurizing implemented in step (c),
- (e) pressurizing the heavier noble gas bound crystal with a noble gas, to a pressure ranging from 100 to 800 MPa, at a temperature ranging above the freezing point of the water in the crystal to room temperature,
- (f) cooling the pressurized heavier noble gas bound crystal to below 120 K, and
- (g) releasing the gas pressure implemented in step (e) and maintaining the temperature of the crystal at cryogenic temperature.
Dependencies are inferred from the claim text.