X-Ray Crystallographic Analysis of Spherical Plant Viruses

烟草坏死病毒是一种由180个拷贝的外壳蛋白和一个单链RNA组成的球形植物病毒。以藜麦为试材，采用差速离心法、凝胶过滤法和蔗糖密度离心法进行提纯。纯化的TNV在SDS-PAGE中仅显示一条M=30,000的条带，在电子显微镜下可见直径约为28 nm的均一颗粒。用0.4M磷酸二氢钠缓冲液，调节pH至6.0，透析制得TNV晶体。TNV晶体以十二面体的形式生长，最大直径为1 mm。晶体的衍射图表明，该晶体属于立方空间群P4_232，a=338&lt；Ang&gt；晶胞中的两个粒子围绕位于(0，0，0)和(1/2，1/2，1/2)处的晶体的主轴相互旋转90。单胞中粒子之间的最近距离为293和。这种晶体对X射线的衍射率达到2.5&lt；Ang&gt；分辨率，辐射损伤很小。三维X射线数据的收集和重原子衍生物的制备正在进行中。

Tobacco necrosis virus (TNV) is a spherical plant virus consisting of 180 copies of coat protein and a single-stranded RNA. TNV was propagated in Chenopodium quinoa, and purified by differential centrifugation, gel filtration and sucrose density centrifugation. The purified TNV showed only one band at M=30,000 in SDS-polyacrylamide gel electrophoresis, and contained homogeneous particles of a diameter of approximately 28 nm in the electron microscope. TNV crystals were produced by dialyzing against 0.4 M sodium phosphate buffer with the pH adjusted to 6.0. The TNV crystals grew in the form of dodecahedron, to a maximum of 1 mm in diameter. The diffraction patterns of the crystals have shown that the crystal belongs to cubic space group p4_232 with a=338 <Ang>. The two particles in the unit cell are rotated 90 to each other about the principal axis of the crystal situated at (0,0,0) and (1/2,1/2,1/2). The nearest distance between the particles in the unit cell is 293 <Ang>. The crystals diffract X-ray to 2.5 <Ang> resolution and showed little radiation damage. Collection of three-dimensional X-ray data and preparation of heavy atom derivatives are in progress.