Open AccessPurification, crystallization and preliminary X‐ray diffraction of SecDF, a translocon‐associated membrane protein, from Thermus thermophilus
Author(s) -
Tsukazaki Tomoya,
Mori Hiroyuki,
Fukai Shuya,
Mori Yusuke,
Numata Tomoyuki,
Perederina Anna,
Adachi Hiroaki,
Matsumura Hiroyoshi,
Takano Kazufumi,
Murakami Satoshi,
Inoue Tsuyoshi,
Sasaki Takatomo,
Vassylyev Dmitry G.,
Nureki Osamu,
Ito Koreaki
Publication year - 2006
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309106007779
Subject(s) - thermus thermophilus , translocon , crystallization , escherichia coli , crystallography , membrane , periplasmic space , chemistry , membrane protein , materials science , biochemistry , organic chemistry , gene
Thermus thermophilus has a multi‐path membrane protein, TSecDF, as a single‐chain homologue of Escherichia coli SecD and SecF, which form a translocon‐associated complex required for efficient preprotein translocation and membrane‐protein integration. Here, the cloning, expression in E. coli , purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in the presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 Å upon X‐ray irradiation, revealing that they belonged to space group P 4 3 2 1 2. Attempts were made to improve the diffraction quality of the crystals by combinations of micro‐stirring, laser‐light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 Å resolution and preliminary success in the single‐wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three‐dimensional structure of this important membrane component of the protein‐translocation machinery.