Sumida Tomomi, Yanagisawa Tatsuo, Ishii Ryohei, Yokoyama Shigeyuki
RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Sep 1;66(Pt 9):1115-8. doi: 10.1107/S1744309110032008. Epub 2010 Aug 28.
GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, was overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The GenX-LysAMS crystals belonged to the triclinic space group P1, with unit-cell parameters a=54.80, b=69.15, c=94.08 A, alpha=95.47, beta=106.51, gamma=90.46 degrees, and diffracted to 1.9 A resolution. Furthermore, GenX was cocrystallized with translation elongation factor P (EF-P), which is believed to be a putative substrate of GenX, and LysAMS using PEG 4000 and ammonium sulfate as precipitants. The GenX-EF-P-LysAMS crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a=105.93, b=102.96, c=119.94 A, beta=99.4 degrees, and diffracted to 2.5 A resolution. Structure determination of the E. coli GenX-LysAMS and GenX-EF-P-LysAMS complexes by molecular replacement was successful and structure refinements are now in progress.
GenX是大肠杆菌中的一种赖氨酰 - tRNA合成酶旁系同源物，在大肠杆菌中过表达，通过三步色谱法纯化，并使用聚乙二醇4000作为沉淀剂，通过悬滴气相扩散法与赖氨酰腺苷酸类似物（LysAMS）共结晶。GenX - LysAMS晶体属于三斜晶系空间群P1，晶胞参数为a = 54.80，b = 69.15，c = 94.08 Å，α = 95.47，β = 106.51，γ = 90.46°，衍射分辨率达到1.9 Å。此外，GenX与翻译延伸因子P（EF - P）共结晶，EF - P被认为是GenX的一种假定底物，同时还与LysAMS共结晶，使用聚乙二醇4000和硫酸铵作为沉淀剂。GenX - EF - P - LysAMS晶体属于单斜晶系空间群P2(1)，晶胞参数为a = 105.93，b = 102.96，c = 119.94 Å，β = 99.4°，衍射分辨率达到2.5 Å。通过分子置换法成功测定了大肠杆菌GenX - LysAMS和GenX - EF - P - LysAMS复合物的结构，目前正在进行结构优化。
