1	Automatic Protein Crystallization in an Anaerobic Environment Bret Dillard B.C. Wang Lab University of Georgia 07/21/06
2	Basis for Anaerobic Crystallization Many proteins exist in an anaerobic environment Lose cofactors such as iron-sulfur clusters in oxygen environment Need to reproduce redox state of cofactors such as FAD⁺ and NAD⁺ Many crystallographers need to see different redox states of protein conformations for biological studies
3	Many Ways to Solve This Problem Anaerobic experiments (in a chamber) Sitting drop experiments Hanging drop experiments Capillary diffusion experiments Or use Douglas robot in Bactron X chamber
4	Differences Between Techniques Amount of Protein Hanging drop, sitting drop, and capillary techniques use copious amounts of protein and crystallizing agent Microbatch diffusion uses 300nl of protein per condition (in this experiment) Microbatch allows use of same mother liquor for many experiments
5	The Chamber Using Sheldon Manufacturing Bactron X chamber with custom wall joint one can run robot with computer outside the chamber
6	Connections and Oxygen Detection 24 parallel ports allow computer to run robot from outside the chamber Using 5% hydrogen / 95% nitrogen gas mix we are allowed to detect oxygen levels inside the chamber The same gas mix also allows us to use palladium catalyst to remove oxygen at an accelerated rate
7	The Overall Setup
8	Crystallization of Oxygen Sensitive Protein We have purified and crystallized four proteins that are not stable in an oxygen environment The structure of one of these proteins (rubrerythrin) has been solved using anaerobic techniques as of this moment. This protein was previously solved with zinc being coordinated in an aerobic environment The native protein contains iron but is unstable in oxygen We now have the native iron form of this oxygen labile protein through using this system
9	Iron Form of Rubrerythrin
10	Advantages of Method Use of less protein Sitting drop, hanging drop, and capillary methods use large volumes of protein Microbatch uses only 300nl of protein per condition Less time involved in experiment Sitting drop, hanging drop, and capillary methods require intensive attention per experiment Microbatch requires only initial setup and the robot performs the experiment