Kong L B, Siva A C, Kickhoefer V A, Rome L H, Stewart P L
Department of Molecular and Medical Pharmacology, Crump Institute for Biological Imaging, University of California at Los Angeles School of Medicine, 90095-1770, USA.
RNA. 2000 Jun;6(6):890-900. doi: 10.1017/s1355838200000157.
The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus. To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-A-resolution structure. Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined "barrel only" images indicate a possible symmetry mismatch between the barrel and the caps. The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap.
穹窿体复合物是一种普遍存在的13兆道尔顿核糖核蛋白组装体，由三种在真核生物中高度保守的蛋白质（TEP1，240千道尔顿；VPARP，193千道尔顿；MVP，100千道尔顿）和一种非翻译RNA（vRNA）组成。已证明穹窿体可影响癌细胞的多药耐药性，其中一个特定成分MVP被认为在药物从细胞核的转运中起作用。为了确定vRNA的位置，用核糖核酸酶处理穹窿体，并对所得复合物进行冷冻电子显微镜（cryo-EM）分析。使用单颗粒重建技术，将3476个颗粒图像合并以生成分辨率为22埃的结构。核糖核酸酶处理后的穹窿体与先前计算的完整穹窿体重建之间的差异图谱显示，vRNA位于穹窿体帽的末端。在这个位置，vRNA可能与穹窿体的内部和外部环境相互作用。在帽顶部发现的16倍密度环使得能够在冷冻电子显微镜穹窿体密度内对穹窿体TEP1蛋白的WD40重复结构域进行建模。化学计量学考虑以及对计算选择和细化的“仅桶状”图像更高分辨率的发现均表明桶状结构和帽之间可能存在对称性不匹配。该复合物的分子结构正在显现，96个MVP拷贝组成八重对称的桶状结构，vRNA与一个TEP1拷贝以及四个预测的VPARP拷贝共同构成每个帽。
