期刊
JOURNAL OF CRYSTAL GROWTH
卷 312, 期 5, 页码 714-719出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jcrysgro.2009.11.060
关键词
Biocrystallization; Crystal morphology; X-ray diffraction; Single crystal growth; Proteins; Ionic liquids
资金
- Portuguese Science and Technology Foundation (FCT-MCTES) [POCI/QUI/57641/2004]
- FEDER
- [SFRH/BD/37948/2007]
- Fundação para a Ciência e a Tecnologia [SFRH/BD/37948/2007, POCI/QUI/57641/2004] Funding Source: FCT
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.
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