The use of ionic liquids as crystallization additives allowed to overcome nanodrop scaling up problems: A success case for producing diffraction-quality crystals of a nitrate reductase
The use of ionic liquids as crystallization additives allowed to overcome nanodrop scaling up problems: A success case for producing diffraction-quality crystals of a nitrate reductase
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DOI：
10.1016/j.jcrysgro.2009.11.060
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发表时间：
2010-02-15
影响因子：
1.8
通讯作者：
Romao, Maria Joao
中科院分区：
文献类型：
--
作者：
Coelho, Catarina;Trincao, Jose;Romao, Maria Joao
利用在格勒诺布尔EMBL的机器人设施获得的一个单晶，以1.5埃分辨率解析了来自钩虫贪铜菌（C）的异二聚体周质硝酸还原酶（NapAB）的天然结构。这个家族的蛋白质的反应机制最近进行了修订，根据新的晶体学证据，需要新的结构研究，以澄清这一新的机制的影响。几个纳米滴结晶试验产生了Cnecator NapAB的微晶。然而，扩大规模的尝试系统地失败了，没有产生任何合适的晶体。只有使用离子液体（IL），我们才能够以可重复的方式生长更大的晶体，其衍射X射线的分辨率为1.7埃。通过使用IL [C（4）mim] Cl作为结晶添加剂，我们获得了获得良好质量晶体的再现性。虽然没有IL分子可以确定在电子密度图中，在IL的存在和不存在下生长的晶体有很大的差异，在细胞常数。这是第一次报告使用IL的困难结晶问题。现在报道的程序可用于晶体优化，例如尺寸增加或细针的改进，以及将结晶条件从纳升扩大到微升滴体积。(C)2009爱思唯尔有限公司版权所有。
The native structure of the heterodimeric periplasmic nitrate reductase (NapAB) from Cupriavidus (C) necator was solved at 1.5 angstrom resolution, using one single crystal obtained at the robot facility at the EMBL, Grenoble. The reaction mechanism for this family of proteins was recently revised, based on new crystallographic evidence, and new structural studies are required to clarify this new mechanistic implication. Several nanodrop crystallization trials yielded microcrystals of the C necator NapAB. However, scale-up attempts systematically failed and did not yield any suitable crystals. Only with the use of ionic liquids (IL) were we able to grow, in a reproducible manner, larger crystals, which diffracted X-rays to 1.7 angstrom resolution. By using the IL [C(4)mim]Cl as a crystallization additive, we achieved reproducibility in obtaining good quality crystals. Although no IL molecules could be identified in the electron density maps, the crystals grown in the presence and absence of IL have large differences in cell constants. This is the first report of the use of IL for a difficult crystallization problem. The procedure now reported can be applied for crystal optimization such as size increase or improvement of fine needles, as well as for scaling-up crystallization conditions from nanolitre to microlitre drop volumes. (C) 2009 Elsevier B.V. All rights reserved.