Hagelueken Gregor, Huang Hexian, Harlos Karl, Clarke Bradley R, Whitfield Chris, Naismith James H
Biomedical Sciences Research Complex, The University of St Andrews, North Haugh, St Andrews KY16 9ST, Scotland.
Acta Crystallogr D Biol Crystallogr. 2012 Oct;68(Pt 10):1371-9. doi: 10.1107/S0907444912029599. Epub 2012 Sep 18.
WbdD is a bifunctional kinase/methyltransferase that is responsible for regulation of lipopolysaccharide O antigen polysaccharide chain length in Escherichia coli serotype O9a. Solving the crystal structure of this protein proved to be a challenge because the available crystals belonging to space group I23 only diffracted to low resolution (>95% of the crystals diffracted to resolution lower than 4 Å and most only to 8 Å) and were non-isomorphous, with changes in unit-cell dimensions of greater than 10%. Data from a serendipitously found single native crystal that diffracted to 3.0 Å resolution were non-isomorphous with a lower (3.5 Å) resolution selenomethionine data set. Here, a strategy for improving poor (3.5 Å resolution) initial phases by density modification and cross-crystal averaging with an additional 4.2 Å resolution data set to build a crude model of WbdD is desribed. Using this crude model as a mask to cut out the 3.5 Å resolution electron density yielded a successful molecular-replacement solution of the 3.0 Å resolution data set. The resulting map was used to build a complete model of WbdD. The hydration status of individual crystals appears to underpin the variable diffraction quality of WbdD crystals. After the initial structure had been solved, methods to control the hydration status of WbdD were developed and it was thus possible to routinely obtain high-resolution diffraction (to better than 2.5 Å resolution). This novel and facile crystal-dehydration protocol may be useful for similar challenging situations.
WbdD是一种双功能激酶/甲基转移酶，负责调控大肠杆菌O9a血清型中脂多糖O抗原多糖链的长度。解析该蛋白质的晶体结构颇具挑战，因为属于I23空间群的现有晶体仅能衍射到低分辨率（超过95%的晶体衍射分辨率低于4 Å，大多数仅为8 Å），且它们是非同晶型的，晶胞尺寸变化超过10%。从偶然发现的一个衍射到3.0 Å分辨率的天然单晶获得的数据与较低分辨率（3.5 Å）的硒代甲硫氨酸数据集非同晶型。本文描述了一种通过密度修正和与额外的4.2 Å分辨率数据集进行交叉晶体平均来改善较差（3.5 Å分辨率）初始相位以构建WbdD粗模型的策略。使用该粗模型作为掩模来裁剪3.5 Å分辨率的电子密度，成功得到了3.0 Å分辨率数据集的分子置换解。所得图谱用于构建WbdD的完整模型。单个晶体的水合状态似乎是WbdD晶体衍射质量变化的基础。在解析出初始结构后，开发了控制WbdD水合状态的方法，从而能够常规获得高分辨率衍射（分辨率优于2.5 Å）。这种新颖且简便的晶体脱水方案可能对类似的具有挑战性的情况有用。
