THE SIR SUPPLIED DR. WARD WITH 700MG TE-METHIONINE. TE-METHIONINE WILL BE USED TO SUPPLY REQUIRED METHIONINE FOR THE MET AUXOTROPHIC STRAIN OF E.COLI CONTAINING RECOMBINANT GENES FOR BOTH AEQUORIN AND GFP. THIS WILL ALLOW THESE STRAINS TO EXPRESS AEQUORIN AND GFP WHICH CONTAIN TE-MET AT POSITIONS NORMALLY CONTAINING S-MET IN THE NATIVE PROTEINS. BECAUSE TE-MET IS SENSITIVE TO OXIDATION, WE WILL CONDUCT ALL PROTEIN PURIFICATION AND CRYSTALLIZATION EXPERIMENTS EITHER IN AN INERT (AR) ATMOSPHERE AND/OR UNDER ANOXYGENIC CONDITIONS, USING PROCEDURES FORMALLY CONDUCTED IN OUR LABORATORY. SINGLE-CRYSTAL X-RAY DIFFRACTION DATA WILL BE COLLECTED FROM THESE TE-MET CONTAINING FORMS OF AEQUORIN AND GFP CRYSTALS USING CU-K ALPHA RADIATION SUPPLIED BY A HIGH INTENSITY ROTATING ANODE SOURCE IN OUR LABORATORY. WE ANTICIPATE THAT THESE DATA WILL PROVIDE SIGNALS ALLOWING US TO USE SINGLE ISOMORPHOUS COLLECTION TIME ON THE CHESS SYNCHROTRON LIGHT SOURCE AT CORNELL UNIVERSITY. USING EITHER THE F1 OR F2 BEAM LINE THERE, WE WILL COLLECTED MULTIPLE WAVELENGTH ANOMALOUS DISPERSION DATA AND/OR WAVELENGTH OPTIMI ZED DATA, BY TURNING THE BEAM ENERGY TO OPTIMIZED THE ANOMALOUS DIFFRACTION SIGNAL FROM TELLURIUM. IN THIS WAY WE BELIEVE WE CAN DERIVE HIGH QUALITY PHASE INFORMATION FOR THE DATA, AND THUS ARRIVE AT A HIGH QUALITY, READILY INTERPRETABLE ELECTRON DENSITY MAPS, LEADING TO ACCURATE ATOMIC MODELS FOR BOTH OF THESE PROTEINS.
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