Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Protein Expression Laboratory, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
J Struct Biol. 2014 Mar;185(3):250-6. doi: 10.1016/j.jsb.2013.12.004. Epub 2013 Dec 15.
In the final stage of radiation damage in cryo-electron microscopy of proteins, bubbles of hydrogen gas are generated. Proteins embedded in DNA bubble sooner than free-standing proteins and DNA does not bubble under the same conditions. These properties make it possible to distinguish protein from DNA. Here we explored the scope of this technique ("bubblegram imaging") by applying it to bacteriophage T7, viewed as a partially defined model system. T7 has a thin-walled icosahedral capsid, 60 nm in diameter, with a barrel-shaped protein core under one of its twelve vertices (the portal vertex). The core is densely wrapped with DNA but details of their interaction and how their injection into a host bacterium is coordinated are lacking. With short (10 s) intervals between exposures of 17 electrons/Å(2) each, bubbling starts in the third exposure, with 1-4 bubbles nucleating in the core: in subsequent exposures, these bubbles grow and merge. A 3D reconstruction from fifth-exposure images depicts a bipartite cylindrical gas cloud in the core. In its portal-proximal half, the axial region is gaseous whereas in the portal-distal half, it is occupied by a 3 nm-wide dense rod. We propose that they respectively represent core protein and an end of the packaged genome, poised for injection into a host cell. Single bubbles at other sites may represent residual scaffolding protein. Thus, bubbling depends on dose rate, protein amount, and tightness of the DNA seal.
在蛋白质冷冻电子显微镜辐射损伤的最后阶段，会产生氢气气泡。嵌入在 DNA 中的蛋白质比独立的蛋白质更早地起泡，并且在相同条件下 DNA 不会起泡。这些特性使得蛋白质和 DNA 可以区分开来。在这里，我们通过将其应用于噬菌体 T7 来探索该技术（“气泡图成像”）的应用范围，将其视为部分定义的模型系统。T7 具有薄壁二十面体衣壳，直径为 60nm，在其十二个顶点之一（门户顶点）下有一个桶形蛋白质核心。核心被 DNA 紧密包裹，但它们之间的相互作用以及如何协调它们注入宿主细菌的细节尚不清楚。每次曝光间隔 17 电子/Å(2)，曝光时间为 10 秒，第三次曝光时开始起泡，核心中出现 1-4 个气泡核：在随后的曝光中，这些气泡会生长和融合。第五次曝光图像的三维重建描绘了核心中的二分柱形气云。在其门户近端的一半中，轴向区域是气态的，而在门户远端的一半中，它被一个 3nm 宽的密集棒占据。我们提出它们分别代表核心蛋白和包装基因组的末端，准备注入宿主细胞。其他部位的单个气泡可能代表残留的支架蛋白。因此，起泡取决于剂量率、蛋白质含量和 DNA 密封的紧密程度。
