Crystal structure of the YjgF/YER057c/UK114 family protein from the hyperthermophilic Archaeon Sulfolobus tokodaii strain 7
Crystal structure of the YjgF/YER057c/UK114 family protein from the hyperthermophilic Archaeon Sulfolobus tokodaii strain 7
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DOI：
10.1002/prot.20778
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发表时间：
2006-02-01
影响因子：
2.9
通讯作者：
Tanokura, M
中科院分区：
文献类型：
--
作者：
Miyakawa, T;Lee, WC;Tanokura, M
材料和方法。蛋白质生产、结晶和 X 射线衍射数据收集。如所述进行蛋白质表达、纯化和结晶。 16 通过坐滴气相扩散法在 293 K 下 3 天内获得晶体。储库溶液使用 16%(w/v) PEG 10,000、0.1 M Bis-Tris (pH 5.3) 和 0.1 M 醋酸铵。将晶体转移到含有 18%(w/v) PEG 10,000 作为沉淀剂、0.12 M Bis-Tris (pH 5.3)、0.12 M 乙酸铵和 20%(v/v) 甘油的冷冻保护剂溶液中，然后在氮气流中快速冷却。使用安装在 Rigaku FR-E 旋转阳极 X 射线发生器（Rigaku，日本）上的 R-AXIS VII 图像板探测器，使用操作软件 CrystalClear (Rigaku/MSC) 在 100 K 下收集衍射数据，并使用 MOSFLM17 和 SCALA 进行处理。 18 结构测定。使用CCP4 suites19的程序MOLREP和枯草芽孢杆菌YabJ的坐标（PDB代码1QD9，与ST0811有50%序列同一性）通过分子替换方法解析了ST0811的结构。 12 MOLREP 使用空间群 R3 中分辨率范围为 19.0–2.0 Å 的数据运行。百分之五的反射被排除在与 Rfree 值交叉验证的总数之外。刚体模型最初使用 Refmac5、20 程序进行细化，然后使用 Xtal-View21 和 Refmac5 执行多个手动模型重建和模型细化周期。 ARP/wARP 程序自动拾取水分子22，然后根据 Fo-Fc 和 2Fo-Fc 图谱中的峰高和距离标准进行确认。使用 PROCHECK 评估模型的质量。 23 坐标已存入蛋白质数据库，登录号为 1X25。
Materials and Methods. Protein production, crystallization and X-ray diffraction data collection. Protein expression, purification, and crystallization were performed as described. 16 The crystals were obtained by the sitting-drop vapor-diffusion method at 293 K in 3 days. The reservoir solution used 16%(w/v) PEG 10,000, 0.1 M Bis-Tris (pH 5.3), and 0.1 M ammonium acetate. Crystals were transferred into the cryoprotectant solution containing 18%(w/v) PEG 10,000 as precipitant, 0.12 M Bis-Tris (pH 5.3), 0.12 M ammonium acetate, and 20%(v/v) glycerol and were then flash-cooled in a nitrogen stream. Diffraction data were collected at 100 K with an R-AXIS VII image plate detector mounted on a Rigaku FR-E rotating-anode X-ray generator (Rigaku, Japan) using the operation software CrystalClear (Rigaku/MSC) and were processed with MOSFLM17 and SCALA. 18 Structure determination. The structure of the ST0811 was solved by the molecular replacement method using the program MOLREP of the CCP4 suites19 and the coordinates of Bacillus subtilis YabJ (PDB code 1QD9, 50% sequence identity to ST0811). 12 MOLREP was run using the data with a resolution range of 19.0–2.0 Å in the space group R3. Five percent of the reflections were excluded from the total for cross-validation with the Rfree value. The rigid-body model was initially refined with the program Refmac5, 20 and several cycles of manual model rebuilding and model refinement were then performed using Xtal-View21 and Refmac5. Water molecules were automatically picked up by the ARP/wARP program, 22 and they were then confirmed based on peak heights and distance criteria in the Fo-Fc and 2Fo-Fc maps. The quality of the model was evaluated with PROCHECK. 23 The coordinates have been deposited into the Protein Data Bank with the accession number 1X25.