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嗜热栖热菌RadA内含肽的克隆、表达、纯化、结晶及初步X射线衍射数据

Cloning, expression, purification, crystallization and preliminary X-ray diffraction data of the Pyrococcus horikoshii RadA intein.

作者信息

Lyskowski Andrzej, Oeemig Jesper S, Jaakkonen Anniina, Rommi Katariina, DiMaio Frank, Zhou Dongwen, Kajander Tommi, Baker David, Wlodawer Alexander, Goldman Adrian, Iwaï Hideo

机构信息

Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, PO Box 65, Helsinki FIN-00014, Finland.

出版信息

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 May 1;67(Pt 5):623-6. doi: 10.1107/S1744309111008372. Epub 2011 Apr 28.

Abstract

The RadA intein from the hyperthermophilic archaebacterium Pyrococcus horikoshii was cloned, expressed and purified for subsequent structure determination. The protein crystallized rapidly in several conditions. The best crystals, which diffracted to 1.75 Å resolution, were harvested from drops consisting of 0.1 M HEPES pH 7.5, 3.0 M NaCl and were cryoprotected with Paratone-N before flash-cooling. The collected data were processed in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 58.1, b = 67.4, c = 82.9 Å. Molecular replacement with Rosetta using energy- and density-guided structure optimization provided the initial solution, which is currently under refinement.

摘要

克隆、表达并纯化了嗜热古细菌火球菌(Pyrococcus horikoshii)的RadA内含肽,用于后续的结构测定。该蛋白在几种条件下迅速结晶。衍射分辨率达到1.75 Å的最佳晶体是从含有0.1 M HEPES pH 7.5、3.0 M NaCl的液滴中收获的,并在快速冷却前用Paratone-N进行冷冻保护。收集到的数据在正交空间群P2(1)2(1)2(1)中进行处理,晶胞参数为a = 58.1、b = 67.4、c = 82.9 Å。使用Rosetta通过能量和密度引导的结构优化进行分子置换,得到了初始解,目前该解正在优化中。

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