Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography
Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography
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DOI：
10.1128/jb.00747-10
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发表时间：
2010-11-01
影响因子：
3.2
通讯作者：
Horowitz, Mark
中科院分区：
文献类型：
--
作者：
Amat, Fernando;Comolli, Luis R.;Horowitz, Mark
这些细菌和古生菌的表面层（S层）阐述了这些晶体结构，已经研究了40年。然而，大多数结构分析都是基于从细胞中分离的负染色S层片段的电子显微镜，这可能会引入染色伪影并允许易于自组装的结构重排。我们提出了一个定量分析的结构和组织的完整的生长细胞的革兰氏阴性细菌新月柄杆菌使用冷冻电子断层扫描（CET）和统计图像处理的S层。而不是预期的长程秩序，我们观察到不同的区域与六角形组织的亚基表现出短程秩序和广泛的周期性分布。此外，发现堆叠的双层的区域，并且当S层蛋白（RsaA）表达水平通过添加多个rsaA拷贝而升高时，这些区域的程度增加。最后，我们结合了高分辨率的氨基酸残基特异性纳米金标记和CET体积的亚断层图像平均值，以提高我们对线性蛋白质序列与目前可用的2 nm分辨率水平下的结构之间的相关性的理解。结果支持这样的观点，即从负染色断层扫描预测的U形RsaA单体从一个顶点的N末端（对应于3重对称轴）到相对顶点的C末端（形成突出的6重对称轴）。这些信息将有助于未来的努力，分析亚基的相互作用和指导选择的内部网站显示的异源蛋白质片段。
The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.