Localization and orientation of the gamma-tubulin small complex components using protein tags as labels for single particle EM.
Localization and orientation of the gamma-tubulin small complex components using protein tags as labels for single particle EM.
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DOI：
10.1016/j.jsb.2009.08.012
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发表时间：
2009-12
影响因子：
3
通讯作者：
Agard, David A.
中科院分区：
文献类型：
--
作者：
Choy, Rebeca M.;Kollman, Justin M.;Zelter, Alex;Davis, Trisha N.;Agard, David A.
γ-微管蛋白小复合物（γ-TuSC）是真核生物中普遍保守的含有微管（MT）成核蛋白：γ-微管蛋白的复合物。γ-TuSC是一种异源四聚体，具有γ-微管蛋白的两个拷贝，Spc98p和Spc97p各一个拷贝。以前，γ-TuSC的结构是用25 A分辨率的单粒子电子显微镜（EM）测定的。γ-TuSC呈Y形，具有单一的柔性臂，这可能是调节MT成核的关键。EM金标显示了γ-微管蛋白在y顶部的位置。体内荧光共振能量转移（FRET）提示了Spc98p和Spc97p的相对方向，但没有区分哪一个大亚基构成了柔性臂。在这里，我们使用荧光蛋白作为共价附着的标签，使用类平均和三维随机锥形倾斜重建来确认体内FRET结果，清楚地表明Spc98p/97p c -末端直接与γ-微管蛋白相互作用。最重要的是，我们确定了柔性臂属于Spc98p，我们的数据还表明，Spc98p和Spc97p的n端是交叉的。更一般地说，我们的结果证实，尽管它们的尺寸小，共价附着的荧光蛋白在单颗粒EM中作为亚基标签表现良好。
γ-Tubulin Small Complex (γ-TuSC) is the universally-conserved complex in eukaryotes that contains the microtubule (MT) nucleating protein: γ-tubulin. γ-TuSC is a heterotetramer with two copies of γ-tubulin and one copy each of Spc98p and Spc97p. Previously, the structure of γ-TuSC was determined by single particle Electron Microscopy (EM) at 25 A resolution. γ-TuSC is Y shaped with a single flexible arm that could be the key to regulating MT nucleation. EM gold labeling revealed the locations of γ-tubulin at the top of the Y. In vivo Fluorescence Resonance Energy Transfer (FRET) suggested the relative orientations of Spc98p and Spc97p but did not distinguish which large subunit formed the flexible arm. Here, using fluorescent proteins as covalently attached tags, we used class averages and 3D random conical tilt reconstructions to confirm the in vivo FRET results, clearly demonstrating that the Spc98p/97p C-termini interact directly with γ-tubulin. Most significantly we have determined that the flexible arm belongs to Spc98p and our data also suggests that the N-termini of Spc98p and Spc97p are crossed. More generally, our results confirm that despite their small size, covalently attached fluorescent proteins perform well as subunit labels in single particle EM.
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