Visualization of ATP synthase dimers in mitochondria by electron cryo-tomography.
Visualization of ATP synthase dimers in mitochondria by electron cryo-tomography.
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DOI：
10.3791/51228
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发表时间：
2014-09-14
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影响因子：
--
通讯作者：
Kühlbrandt W
中科院分区：
文献类型：
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作者：
Davies KM;Daum B;Gold VA;Mühleip AW;Brandt T;Blum TB;Mills DJ;Kühlbrandt W
电子冷冻断层扫描是结构生物学中的一种强大工具，能够可视化生物样品的三维结构，例如细胞、细胞器、膜囊泡或病毒的分子细节。为了实现这一点，含水样品在液态乙烷中快速玻璃化，这将其保持在接近天然的冷冻水合状态。在电子显微镜中，在液氮温度下记录倾斜序列，从中重建3D断层图像。断层体积的信噪比本来就很低。可识别的，经常性的功能增强subtomograms平均，其中个别的子体积被切断，对齐和平均，以减少噪音。通过这种方式，可以获得分辨率为2nm或更好的3D图。将可用的高分辨率结构与3D体积相匹配，然后在其天然环境中产生蛋白质复合物的原子模型。在这里，我们展示了我们如何使用电子冷冻断层扫描研究在线粒体中的大型膜蛋白复合物的原位组织。我们发现，ATP酶组织在行的二聚体沿着高度弯曲的顶点的内膜嵴，而复杂的I是随机分布在膜区域的行的两侧。通过亚断层平均，我们获得了嵴膜内线粒体ATP合酶二聚体的结构。
Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.