Wery J P, Reddy V S, Hosur M V, Johnson J E
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392.
J Mol Biol. 1994 Jan 14;235(2):565-86. doi: 10.1006/jmbi.1994.1014.
The structure of the black beetle nodavirus has been refined at 2.8 A resolution by alternate use of restrained least-squares atomic coordinate refinement and phase refinement by real space averaging with the 5-fold non-crystallographic symmetry in the crystal. The coordinates were also refined by simulated annealing. The final R-factor for all data with I/sigma(I) > 4 was 22.1%. A total of 7692 atoms were refined in one icosahedral asymmetric unit which included 273 oxygen atoms of ordered water molecules. Three identical gene products of 407 amino acids form one icosahedral asymmetric unit. Each is located in a structurally unique position, identified as A, B or C, consistent with a T = 3 quasi equivalent lattice. Icosahedral pentamers are formed by A subunits while B and C subunits alternate about icosahedral 3-fold axes to form quasi hexamers. Five calcium ions are located within the icosahedral asymmetric unit and stabilize the quasi 3-fold related intersubunit contacts between A, B and C. The final model consists of coordinates for residues 56 to 379 of all three subunits and residues 20 to 31 from the C subunit only. Atom positions for the sugar-phosphate backbone were modeled for ten nucleotides close to the icosahedral 2-fold axes. Symmetry equivalent polyribonucleotides form a helical duplex at each icosahedral 2-fold axis. The three subunits display an eight-stranded beta-barrel fold, very similar to the subunit structures observed in most other icosahedral RNA viruses analyzed. Quasi equivalence is regulated by the ordered RNA and residues 20 to 31 in the C subunit to form a "flat inter subunit contact" at icosahedral 2-fold joints. The RNA and polypeptide are disordered at the quasi 2-fold joints and this results in a "bent inter subunit contact". Although similar quaternary structures were seen in T = 3 plant viruses studied, RNA did not play a role as a molecular switch in those structures. The autocatalytic, post assembly, cleavage of the initial gene product at residue Asn363/Ala364 to form a stable and infectious particle is probably the result of an acid catalyzed main-chain hydrolysis in which Asp75 is the proton donor. The reaction is initiated by assembly which places Asp75 in a hydrophobic environment created by quaternary interactions which raises its pK to 5.6. The region in which the reaction occurs is formed by an internal helical bundle that has not been seen in other virus structures.
通过交替使用约束最小二乘原子坐标精修和利用晶体中的 5 重非晶体学对称性进行实空间平均的相位精修，黑甲虫诺达病毒的结构已在 2.8 Å 分辨率下得到精修。坐标也通过模拟退火进行了精修。对于所有 I/σ(I) > 4 的数据，最终的 R 因子为 22.1%。在一个二十面体不对称单元中总共精修了 7692 个原子，其中包括有序水分子的 273 个氧原子。407 个氨基酸的三个相同基因产物形成一个二十面体不对称单元。每个都位于一个结构独特的位置，被确定为 A、B 或 C，与 T = 3 准等效晶格一致。二十面体五聚体由 A 亚基形成，而 B 和 C 亚基围绕二十面体 3 重轴交替形成准六聚体。五个钙离子位于二十面体不对称单元内，稳定 A、B 和 C 之间准 3 重相关的亚基间接触。最终模型由所有三个亚基的 56 至 379 位残基以及仅 C 亚基的 20 至 31 位残基的坐标组成。糖 - 磷酸主链的原子位置针对靠近二十面体 2 重轴的十个核苷酸进行了建模。对称等效的多核糖核苷酸在每个二十面体 2 重轴处形成螺旋双链体。这三个亚基呈现出八链β - 桶状折叠，与在大多数其他已分析的二十面体 RNA 病毒中观察到的亚基结构非常相似。准等效性由有序 RNA 和 C 亚基中的 20 至 31 位残基调节，以在二十面体 2 重连接处形成“平坦的亚基间接触”。RNA 和多肽在准 2 重连接处无序，这导致了“弯曲的亚基间接触”。尽管在研究的 T = 3 植物病毒中观察到了类似的四级结构，但在那些结构中 RNA 并未作为分子开关发挥作用。初始基因产物在 Asn363/Ala364 残基处的自催化、组装后切割以形成稳定且有感染性的颗粒，可能是酸催化主链水解的结果，其中 Asp75 是质子供体。该反应由组装引发，组装将 Asp75 置于由四级相互作用产生的疏水环境中，使其 pK 升高至 5.6。反应发生的区域由一个在其他病毒结构中未见的内部螺旋束形成。
