A GAS CELL FOR COLLECTING X-RAY-DIFFRACTION DATA FROM PROTEINS

An apparatus for varying the partial pressure of gas in equilibrium with a mounted protein crystal and collecting X-ray diffraction data is described. This gas cell allows data sets at several different partial pressures to be collected from the same crystal and is, therefore, well suited for sensitive difference studies. Data have been collected from orthorhombic human hemoglobin A0 crystals at 0 and 406 torr oxygen to a nominal resolution of 2.8 angstrom using Cu Kalpha radiation (1 torr = 133.32 Pa). The crystals were grown in 11% PEG 8000 at 277 K. The crystals were then transferred to 62% PEG 8000 and warmed to 288 K to match the conditions used in spectroscopic studies of single crystals [Mozzarelli, Rivetti, Rossi, Henry & Eaton (1991). Nature (London), 351, 416-4191. The measured time constant for oxygen binding by hemoglobin in the gas cell was 174 min. A difference experiment conducted on hemoglobin at 0 and 406 torr oxygen pressure yielded data with R(sym)'s of 5.2 and 6.5%, respectively, and an R factor between data sets of 19.5%. Thus, this cell is suitable for determining the structures of partially ligated hemoglobin.