Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus

Tomoya Tsukazaki, Hiroyuki Mori, Shuya Fukai, Tomoyuki Numata, Anna Perederina, Hiroaki Adachi, Hiroyoshi Matsumura, Kazufumi Takano, Satoshi Murakami, Tsuyoshi Inoue, Yusuke Mori, Takatomo Sasaki, Dmitry G. Vassylyev, Osamu Nureki, Koreaki Ito

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Abstract

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 P43212. 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.

Original languageEnglish (US)
Pages (from-to)376-380
Number of pages5
JournalActa Crystallographica Section F: Structural Biology and Crystallization Communications
Volume62
Issue number4
DOIs
StatePublished - Apr 1 2006

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All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Genetics
  • Condensed Matter Physics

Cite this

Tsukazaki, T., Mori, H., Fukai, S., Numata, T., Perederina, A., Adachi, H., Matsumura, H., Takano, K., Murakami, S., Inoue, T., Mori, Y., Sasaki, T., Vassylyev, D. G., Nureki, O., & Ito, K. (2006). Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus. Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 62(4), 376-380. https://doi.org/10.1107/S1744309106007779