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嗜碱诺卡氏菌属菌株F96几丁质酶F1(ChiF1)的结晶及初步X射线晶体学分析

Crystallization and preliminary X-ray crystallographic analysis of chitinase F1 (ChiF1) from the alkaliphilic Nocardiopsis sp. strain F96.

作者信息

Matsui Tsutomu, Kumasaka Takashi, Endo Kimiko, Sato Takao, Nakamura Satoshi, Tanaka Nobuo

机构信息

Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.

出版信息

Acta Crystallogr D Biol Crystallogr. 2004 Nov;60(Pt 11):2016-8. doi: 10.1107/S0907444904020475. Epub 2004 Oct 20.

Abstract

Chitinase F1 (ChiF1) isolated from the alkaliphilic Nocardiopsis sp. strain F96 is a family 18 chitinase that hydrolyzes chitin, an insoluble beta-1,4-linked polymer of N-acetylglucosamine. Crystals of recombinant ChiF1 with molecular weight of 33 000 Da were grown to a suitable size for X-ray structure analysis using 18%(w/v) polyethylene glycol 8000, 200 mM zinc acetate dehydrate and 100 mM sodium cacodylate buffer pH 6.5. Diffraction data were collected at SPring-8 and show that the crystals belong to the trigonal space group P3(1)12 or P3(2)12, with unit-cell parameters a = 56.0, c = 179.5 A, and diffract X-rays beyond 1.2 A resolution. Crystallographic analysis was carried out using the multiwavelength anomalous diffraction (MAD) method using zinc as the anomalous scatter. The binding of Zn atoms was clarified from the Bijvoet and dispersive Patterson functions, which gave prominent zinc-zinc self-vectors on the Harker section.

摘要

从嗜碱诺卡氏菌属菌株F96中分离出的几丁质酶F1(ChiF1)是一种18家族几丁质酶,可水解几丁质,几丁质是N-乙酰葡糖胺的一种不溶性β-1,4-连接聚合物。使用18%(w/v)聚乙二醇8000、200 mM乙酸锌二水合物和100 mM二甲胂酸钠缓冲液(pH 6.5),将分子量为33000 Da的重组ChiF1晶体生长到适合X射线结构分析的大小。在SPring-8收集衍射数据,结果表明晶体属于三方空间群P3(1)12或P3(2)12,晶胞参数a = 56.0,c = 179.5 Å,并且能衍射超过1.2 Å分辨率的X射线。使用锌作为反常散射体,通过多波长反常衍射(MAD)方法进行晶体学分析。从Bijvoet函数和色散帕特森函数中明确了锌原子的结合情况,这两个函数在哈克截面给出了显著的锌-锌自身向量。

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