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首页> 外文期刊>Acta crystallographica. Section F, Structural biology communications >Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli
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Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli

机译：CofB的克隆，表达，纯化，结晶和X射线晶体学分析，CofB是人产肠毒素的大肠杆菌CFA / III的次要菌毛素亚基

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摘要

Colonization factor antigen III (CFA/III) is one of the virulence factors of human enterotoxigenic Escherichia coli (ETEC) that forms the long, thin, proteinaceous fibres of type IV pili through assembly of its major and minor subunits CofA and CofB, respectively. The crystal structure of CofA has recently been reported; however, the lack of structural information for CofB, the largest among the known type IV pilin subunits, hampers a comprehensive understanding of CFA/III pili. In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 angstrom for the wild-type construct and a = b = 103.47, c = 362.08 angstrom for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from similar to 4.0 to similar to 2.0 angstrom. The initial phase was solved by the single-wavelength anomalous dispersion (SAD) method using a dehydrated SeMet CofB crystal, which resulted in an interpretable electron-density map.
机译：定殖因子抗原III（CFA / III）是人肠毒素性大肠杆菌（ETEC）的致病因子之一，可通过组装主要和次要亚基CofA和CofB形成IV型菌毛的长而细的蛋白质纤维。最近已经报道了CofA的晶体结构。但是，缺乏已知的IV型菌毛素亚基中最大的CofB的结构信息，这妨碍了对CFA / III菌毛的全面了解。在这项研究中，克隆，表达，纯化和结晶具有N端截短的野生型CofB的构建体。晶体属于菱形体空间群R32，野生型构建体的晶胞参数a = b = 103.97，c = 364.57埃，SeMet衍生形式的晶胞参数为a = b = 103.47，c = 362.08埃。尽管这些晶体的衍射质量最初非常差，但是晶体的脱水将分辨率极限从大约4.0埃提高到了大约2.0埃。初始阶段是使用脱水的SeMet CofB晶体通过单波长异常色散（SAD）方法求解的，从而得到了可解释的电子密度图。

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