Imaging and three-dimensional reconstruction of chemical groups inside a protein complex using atomic force microscopy.
Imaging and three-dimensional reconstruction of chemical groups inside a protein complex using atomic force microscopy.
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使用原子力显微镜在蛋白质复合物内进行化学基团的成像和三维重建。
DOI：
10.1038/nnano.2014.335
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发表时间：
2015-03
影响因子：
38.3
通讯作者：
中科院分区：
文献类型：
--
作者：
扫描探针显微镜可用于对原子尺度的表面进行成像和化学表征。然而，扫描探针显微镜中局部尖端与样品的相互作用将高分辨率图像限制在表面最上面的原子层，，，，，，，，，并且表征材料和生物分子的内部结构对于此类仪器来说是一个挑战。在这里，我们展示了原子力显微镜可用于对蛋白质复合物内部的化学基团进行成像和三维重建。我们使用短单链 DNA 作为成像标签，连接到蛋白质复合物内的目标区域，并使用互补探针 DNA 功能化的 T 形原子力显微镜悬臂梁，使标签能够以序列特异性和亚纳米分辨率定位。在测量标签之间的成对距离后，我们使用简单的几何计算重建了蛋白质复合物内目标化学基团形成的三维结构。生物素-链霉亲和素复合物的实验表明，预测的生物素羧酸基团的三维位点在相应晶体结构中各自位点的 2 Å 范围内，这表明扫描探针显微镜可以补充现有的结构生物学技术，以解决由于其尺寸和复杂性而难以研究的结构。
Scanning probe microscopes can be used to image and chemically characterize surfaces down to the atomic scale,,. However, the localized tip–sample interactions in scanning probe microscopes limit high-resolution images to the topmost atomic layer of surfaces,,,,,,,,, and characterizing the inner structures of materials and biomolecules is a challenge for such instruments. Here, we show that an atomic force microscope can be used to image and three-dimensionally reconstruct chemical groups inside a protein complex. We use short single-stranded DNAs as imaging labels that are linked to target regions inside a protein complex, and T-shaped atomic force microscope cantilevers,functionalized with complementary probe DNAs allow the labels to be located with sequence specificity and subnanometre resolution. After measuring pairwise distances between labels, we reconstruct the three-dimensional structure formed by the target chemical groups within the protein complex using simple geometric calculations. Experiments with the biotin–streptavidin complex show that the predicted three-dimensional loci of the carboxylic acid groups of biotins are within 2 Å of their respective loci in the corresponding crystal structure, suggesting that scanning probe microscopes could complement existing structural biological techniques in solving structures that are difficult to study due to their size and complexity.
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