Inert crystal delivery medium for serial femtosecond crystallography

Patent metadata

Field	Value
Publication number	US-10775322-B2
Application number	US-201615183149-A
Country	US
Kind code	B2
Filing date	Jun 15, 2016
Priority date	Jun 16, 2015
Publication date	Sep 15, 2020
Grant date	Sep 15, 2020

Abstract

A system and method for preparing a crystal delivery medium comprising agarose for serial femtosecond crystallography and uses thereof.

First claim

We claim:

1. A method for preparation of an inert crystal delivery medium, the method comprising the steps of: preparing an agarose solution comprising agarose, a cryoprotectant, and a first crystallization buffer; preparing a crystal solution comprising a plurality of ordered and symmetrically packed membrane-bound protein crystals and a second crystallization buffer, wherein membrane-bound protein of the membrane-bound protein crystals is insoluble in water; and mixing the crystal solution and the agarose solution such that the plurality of membrane-bound protein crystals are suspended within the agarose solution to form an inert crystal delivery medium comprising the plurality of membrane-bound protein crystals, 5.5%-10% (w/v) agarose, the cryoprotectant, the first crystallization buffer, and the second crystallization buffer.

2. The method of claim 1 , wherein the crystal solution is contained within a first syringe, the agarose solution is contained within a second syringe, and the first and second syringes are coupled to mix the crystal solution and agarose solution and, thereby, form the inert crystal delivery medium.

3. The method of claim 1 , wherein the cryoprotectant is provided at a concentration sufficient to prevent formation of ice.

4. The method of claim 1 wherein the first crystallization buffer of the agarose solution and the second crystallization buffer of the crystal solution are the same crystallization buffer.

5. The method of claim 1 , wherein the plurality of the membrane-bound protein crystals comprises crystals of between 100 nm to 40 μm in size.

6. The method of claim 5 , wherein the crystals are between 1 to 10 μm in size.

7. A crystal delivery medium, the medium comprising:
ordered and symmetrically packed membrane-bound protein crystals, wherein membrane-bound protein of the membrane-bound protein crystals is insoluble in water;
5.5%-10% (w/v) agarose;
a cryoprotectant; and
a crystallization buffer.

8. The medium of claim 7 , wherein the cryoprotectant is a polyethylene glycol.

9. The medium of claim 7 , wherein the cryoprotectant is a sugar.

10. The medium of claim 7 , wherein the cryoprotectant is provided at a concentration sufficient to prevent formation of ice.

11. The medium of claim 7 , wherein the cryoprotectant is selected from the group consisting of polyethylene glycols, glycerol, ethylene glycol, non-volatile organics, osmolytes, polyols, sugars, oils, and mixtures thereof.

12. The medium of claim 11 , wherein the cryoprotectant is selected from the group consisting of polyethylene glycols, glycerol, ethylene glycol, sugars, and mixtures thereof.

13. A system for preparation of a crystal delivery medium for serial femtosecond crystallography, the system comprising: ordered and symmetrically packed membrane-bound protein crystals, wherein the membrane-bound protein of the membrane-bound protein crystals is insoluble in water; an agarose solution comprising 5.5%-10% (w/v) agarose, a cryoprotectant, and a crystallization buffer; and a first syringe, a second syringe, and a syringe coupler for mixing the membrane protein crystals into the agarose solution.

14. The system of claim 13 , wherein the cryoprotectant is provided at a concentration sufficient to prevent formation of ice.

Assignees

• Univ Arizona State

Inventors

• Conrad Chelsie
• Fromme Petra
• James Daniel
• Kupitz Christopher

Classifications

• C30B29/58

  Macromolecular compounds · CPC title

• G01N23/2005 Primary

  Preparation of powder samples therefor · CPC title

• G01N33/483

  Physical analysis of biological material · CPC title

• C30B7/14

  the crystallising materials being formed by chemical reactions in the solution · CPC title