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首页> 外文期刊>Journal of Molecular Biology >ELUCIDATION OF CRYSTAL PACKING BY X-RAY DIFFRACTION AND FREEZE-ETCHING ELECTRON MICROSCOPY - STUDIES ON GTP CYCLOHYDROLASE I OF ESCHERICHIA COLI
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ELUCIDATION OF CRYSTAL PACKING BY X-RAY DIFFRACTION AND FREEZE-ETCHING ELECTRON MICROSCOPY - STUDIES ON GTP CYCLOHYDROLASE I OF ESCHERICHIA COLI

机译：X射线衍射和冷冻刻蚀电子显微镜鉴定晶体包装-大肠杆菌的GTP环糖酶I研究。

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A monoclinic crystal modification of GTP cyclohydrolase I (space group P2(1), a = 204.2 Angstrom, b = 210.4 Angstrom, c = 71.8 Angstrom, alpha = gamma = 90 degrees, beta = 95.8 degrees) was studied by freeze-etching electron microscopy and by Patterson correlation techniques. The freeze-etched samples were either shadowed with Pt/C or decorated with monolayers of gold, silver or platinum. Correlation averaged electron micrographs of decoration replicas indicated 5-fold molecular symmetry. In conjunction with the molecular mass of the active GTP cyclohydrolase I enzyme complex of about 210,000 Da, which had been reported in the literature, and a molecular mass of the protomers of 24,700 Da, the electron microscopic observation suggests that the enzyme is a decamer with 5-fold symmetry. The processed images of decorated crystal surfaces also showed that the four protein multimers in the crystal unit cell are related by 4-fold pseudosymmetry. A Patterson analysis of the X-ray data showed two non-crystallographic 5-fold axes, inclined at 12 degrees to each other, thus confirming and extending the electron microscopic findings. Additionally, local 2-fold axes were found in planes perpendicular to the 5-fold particle axes. Thus, the combined X-ray and electron microscope data indicate that GTP cyclohydrolase I is a decamer with D-5 symmetry. A procedure for hkl assignments of the crystal planes observed in electron micrographs was developed. On this basis, it was possible to determine the approximate molecular positions in the ab plane. Independent information on the crystal packing was obtained by single isomorphous replacement and electron density averaging. The 5-fold averaged 6 Angstrom electron density shows that the GTP cyclohydrolase I decamer is torus-shaped with an approximate diameter of 100 Angstrom and a thickness of 65 Angstrom. The study demonstrates that the combination of freeze-etching electron microscopy with Patterson analysis of X-ray data is a powerful approach for the solution of complex crystallographic problems. The procedure for this analysis as well as possible pitfalls are discussed in detail. (C) 1995 Academic Press Limited [References: 24]
机译：通过冷冻蚀刻电子研究了GTP环水解酶I（空间群P2（1），a = 204.2埃，b = 210.4埃，c = 71.8埃，α=伽马= 90度，β= 95.8度）的单斜晶体修饰显微镜和通过帕特森相关技术。冷冻蚀刻的样品用Pt / C遮蔽或用金，银或铂的单层装饰。装饰复制品的相关平均电子显微照片显示5倍分子对称性。结合文献中报道的活性GTP环水解酶I酶复合物的分子量约为210,000 Da，以及原启动子的分子量为24,700 Da，电子显微镜观察表明该酶是5倍对称。装饰的晶体表面的处理图像还显示，晶体单位晶格中的四个蛋白质多聚体与4倍假对称相关。 X射线数据的帕特森分析显示，两个非晶体5折轴彼此倾斜12度，因此证实并扩展了电子显微镜的发现。此外，在垂直于5倍粒子轴的平面中发现了局部2倍轴。因此，结合的X射线和电子显微镜数据表明GTP环水解酶I是具有D-5对称性的十聚体。开发了在电子显微照片中观察到的晶面hkl分配的程序。在此基础上，有可能确定ab平面中的近似分子位置。通过单同晶置换和平均电子密度获得有关晶体堆积的独立信息。 5倍平均6埃电子密度表明，GTP环水解酶I decamer为圆环形，直径约为100埃，厚度为65埃。研究表明，冷冻蚀刻电子显微镜与X射线数据的Patterson分析相结合是解决复杂晶体学问题的有力方法。详细讨论了此分析的过程以及可能的陷阱。 （C）1995 Academic Press Limited [参考号：24]

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