Refinement of high resolution X-ray structure; the case of Taka-amylase A and Lysozyme

Institute for Protein Research, University of Osaka* Japan Space Forum** Maruwa Food Industries, Inc.*** Japan Aerospace Exploration Agency****
â—‹Akifumi Higashiura* Hiroaki Tanaka** Koji Inaka*** Masaru Sato**** Shigeru Sugiyama*** Sachiko Takahashi** Mari Yamanaka** Moritoshi Motohara**** Satoshi Sano**** Tomoyuki Kobayashi**** Mamoru Suzuki* Tetsuo Tanaka**** Atsushi Nakagawa*

Recently, high brilliance and small divergence synchrotron beam lines, X-ray data collection at low temperature and technical advances in crystallographic analysis have significantly improved the resolution of X-ray crystallography. In this study Taka-amylase A and lysozyme were used as a model protein for high resolution X-ray crystallography. Taka-amylase A crystals were grown in microgravity environment (JAXA-GCF project) and lysozyme crystals were grown in the ground. Data collections were performed using synchrotron radiation from SPring-8 beamline BL12B2, BL44XU and Photon Factory beamline BL-5A. Two data sets were collected for high- and low-resolution data to avoid the saturation of high intensity diffraction. The high-resolution diffraction data of Taka-amylase A were observed to 0.94Å and lysozyme were 0.75Å.The data were integrated, scaled and merged using the DENZO and SCALEPACK programs. Taka-amylase A crystals belong to space group P212121, with the cell dimensions a=50.4Å, b=67.4Å, c=130.5Å and lysozyme crystals were P1, a=26.7Å, b=31.0Å, c=33.7Å, α=89.21, β=72.5, γ=67.7. The overall Rmerge based on intensities for all data of Taka-amylase A was 7.7% with its completeness of 97.6% against data to 1.0Å and lysozyme was 4.7%, 88.0%. Refinement was carried out by SHELX programs. The refinement of Taka-amylase A and lysozyme were proceeding against data to 1.0Å and 0.80Å respectively. An R factor and free-R factor of Taka-amylase A was 13.1%, 16.1% and lysozyme was 12.6%, 14.8%. These high-resolution structures were providing us more reliable geometric and conformational properties of the protein.