Structure determination of a novel protein by sulfur SAD using chromium radiation in combination with a new crystal-mounting method.
A novel and easy crystal-mounting technique was developed for the sulfur SAD method using Cr Kalpha radiation (2.29 A). Using this technique, the cryo-buffer and cryoloop around the protein crystal can be removed before data collection in order to eliminate their X-ray absorption. The superiority and reproducibility of the data sets with this mounting technique were demonstrated using tetragonal hen egg-white lysozyme crystals. The structure of a novel protein, PH1109, from Pyrococcus horikoshii OT3 was solved using this technique. At the wavelength of Cr Kalpha radiation, the anomalous signal |DeltaF|/|F| of PH1109 is expected to be 1.72% as this protein of 144 residues includes four methionines and two cysteines. Sulfur SAD phasing was performed using SHELXD and SHELXE. In the case of the data set obtained using this novel crystal-mounting technique, 54.9% of all residues were built with side chains automatically by RESOLVE. On the other hand, only 16.0% were built with side chains for the data set collected using the standard cryoloop. These results indicated that this crystal-mounting technique was superior to the standard loop-mounting method for the measurement of small anomalous differences at longer wavelength and yielded better results in sulfur-substructure solution and initial phasing. The present study demonstrates that the sulfur SAD method with a chromium source becomes enhanced and more practical for macromolecular structure determination using the new crystal-mounting technique.
Kitago
Yu
Y
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Japan.
Watanabe
Nobuhisa
N
Tanaka
Isao
I
D000042:Absorption; D001426:Bacterial Proteins / Q000737:chemistry*; D002857:Chromium / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry; D043902:Pyrococcus horikoshii / Q000737:chemistry; D013455:Sulfur / Q000737:chemistry*
D001426:Bacterial Proteins; D002857:Chromium; D013455:Sulfur; D009113:Muramidase