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首页> 外文期刊>Acta crystallographica.Section D. Biological crystallography >De novo sulfur SAD phasing of the lysosomal 66.3 kDa protein from mouse.
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De novo sulfur SAD phasing of the lysosomal 66.3 kDa protein from mouse.

机译：66.3新创硫悲伤逐步的溶酶体从鼠标kDa蛋白质。

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摘要

The 66.3 kDa protein from mouse is a soluble protein of the lysosomal matrix. It is synthesized as a glycosylated 75 kDa preproprotein which is further processed into 28 and 40 kDa fragments. Despite bioinformatics approaches and molecular characterization of the 66.3 kDa protein, the mode of its maturation as well as its physiological function remained unknown. Therefore, it was decided to tackle this question by means of X-ray crystallography. After expression in a human fibrosarcoma cell line, the C-terminally His-tagged single-chain 66.3 kDa variant and the double-chain form consisting of a 28 kDa fragment and a 40 kDa fragment were purified to homogeneity but could not be separated during the purification procedure. This mixture was therefore used for crystallization. Single crystals were obtained and the structure of the 66.3 kDa protein was solved by means of sulfur SAD phasing using data collected at a wavelength of 1.9 A on the BESSY beamline BL14.2 of Freie Universitat Berlin. Based on the anomalous signal, a 22-atom substructure comprising 21 intrinsic S atoms and one Xe atom with very low occupancy was found and refined at a resolution of 2.4 A using the programs SHELXC/D and SHARP. Density modification using SOLOMON and DM resulted in a high-quality electron-density map, enabling automatic model building with ARP/wARP. The initial model contained 85% of the amino-acid residues expected to be present in the asymmetric unit of the crystal. Subsequently, the model was completed and refined to an R(free) factor of 19.8%. The contribution of the single Xe atom to the anomalous signal was analyzed in comparison to that of the S atoms and was found to be negligible. This work should encourage the use of the weak anomalous scattering of intrinsic S atoms in SAD phasing, especially for proteins, which require both expensive and time-consuming expression and purification procedures, preventing extensive screening of heavy-atom crystal soaks.
机译：66.3 kDa从鼠标是可溶性蛋白蛋白质的溶酶体矩阵。合成糖化75 kDapreproprotein进一步加工成28和40 kDa碎片。方法和分子的特性66.3 kDa蛋白质,其成熟的模式及其生理功能未知的。通过x射线结晶学的问题。表达一个人纤维肉瘤细胞株,c端His-tagged单链66.3 kDa变体和双链形式组成的28 kDa片段和40 kDa片段净化的同质性,但不能在净化过程中分离。因此混合用于结晶。得到了单晶结构66.3 kDa蛋白质是通过解决硫悲伤的逐步使用收集的数据波长在1.9贝茜beamline BL14.2柏林自由大学的。异常信号,22-atom子结构由21个内在原子和一个Xe原子非常低的入住率被发现和提炼2.4解决使用程序SHELXC / D和夏普。DM导致高质量的电子密度地图,支持自动建模ARP /变形。氨基酸残基预计将出现在不对称的晶体。模型完成并精制R(免费)因素的19.8%。Xe原子的异常信号进行了分析比较的原子和被发现可以忽略不计。使用固有的弱反常散射原子在悲伤的定相,尤其是蛋白质,这既需要昂贵和耗时的表达和纯化过程,防止重原子的广泛筛查水晶浸泡。

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