Structure determination of a novel protein by sulphur SAD using chromium radiation in combination with a new crystal mounting method
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A novel and easy crystal mounting technique was developed for the sulphur SADmethod using chromium Kα radiation (2.29Å). Using this technique, the cryo-buffer andcryoloop around the protein crystal can be removed before data collection to eliminate theirX-ray absorption. The superiority and the reproducibility of the datasets with our mountingtechnique were demonstrated using tetragonal hen egg-white lysozyme crystals. The structureof a novel protein, PH1109, from Pyrococcus horikoshii OT3 was solved using thistechnique. At the wavelength of chromium Kα radiation, anomalous signals, <|ΔF|>/<|F|> ofPH1109 is expected to be 1.72%, as this protein of 144 residues includes 4 methionines and 2cysteines. Sulphur SAD phasing was performed using SHELXD and SHELXE. In the case ofthe dataset obtained using our novel crystal mounting technique, 54.9 % of all residues werebuilt with side-chains automatically by RESOLVE. On the other hand, only 16.0 % were builtwith side-chains for the dataset collected using the standard cryoloop. These results indicatedthat our crystal mounting technique was superior to the standard loop mounting method formeasurement of small anomalous differences at longer wavelength, and yielded better resultsin sulphur substructure solution and initial phasing. The present study demonstrated that thesulphur SAD method with a chromium source becomes enhanced more practical formacromolecular structure determination using our crystal mounting technique.
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