Assignment of individual metal redox states in a metalloprotein by crystallographic refinement at multiple X-ray wavelengths.

Crystals of Fd4 were grown by sitting drop vapour diffusion from a reservoir solution containing 25 % (w/v) of polyethylene glycol 400, 0.05 M cadmium chloride and 0.1 M of sodium acetate/acetic acid buffer, pH 4.6, in a flexible glove box (Coy Laboratories, Grass Lake, MI) at less than 1 ppm of oxygen. 1 μl of protein solution at a concentration of 10 mg·ml was mixed with 1 μl of this reservoir solution and incubated in sitting drop well plates at 293 K. Large single crystals of Fd4 grew within two to three days and were harvested into a solution containing 33 % (w/v) of polyethylene glycol 400, 0.05 M cadmium chloride and 0.1 M sodium acetate/acetic acid buffer, pH 4.6, and flash-cooled in liquid nitrogen. Preliminary tests were conducted at Stanford Synchrotron Radiation Laboratory, Palo Alto, and final data collection was carried out at the EMBL BW7A beam line at the DORIS storage ring, DESY, Hamburg, Germany. After mounting the crystal, a fluorescence scan of the K-edge region of iron (7070 7180 eV) was taken from which the wavelengths for the individual data sets were chosen. Data collection was carried out starting at the highest energy (strongest anomalous signal). At each energy, 180 images with 1° rotation angle per image were collected, covering the same angular range of the same crystal in each case and extending over the resolution range of 50.0 1.8 Å.