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

Research output: Contribution to journalArticle

26 Citations (Scopus)

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

Fingerprint

Thermus thermophilus
Crystallization
purification
X-Ray Diffraction
Escherichia coli
Purification
Membrane Proteins
Irradiation
crystallization
membranes
proteins
X ray diffraction
Crystals
Membranes
Cloning
Dehydration
diffraction
Machinery
irradiation
x rays

All Science Journal Classification (ASJC) codes

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

Cite this

Tsukazaki, Tomoya ; Mori, Hiroyuki ; Fukai, Shuya ; Numata, Tomoyuki ; Perederina, Anna ; Adachi, Hiroaki ; Matsumura, Hiroyoshi ; Takano, Kazufumi ; Murakami, Satoshi ; Inoue, Tsuyoshi ; Mori, Yusuke ; Sasaki, Takatomo ; Vassylyev, Dmitry G. ; Nureki, Osamu ; Ito, Koreaki. / Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus. In: Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 2006 ; Vol. 62, No. 4. pp. 376-380.
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title = "Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus",
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 {\AA} 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 {\AA} 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.",
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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, DG, 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, vol. 62, no. 4, pp. 376-380. https://doi.org/10.1107/S1744309106007779

Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus. / Tsukazaki, Tomoya; Mori, Hiroyuki; Fukai, Shuya; Numata, Tomoyuki; Perederina, Anna; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo; Vassylyev, Dmitry G.; Nureki, Osamu; Ito, Koreaki.

In: Acta Crystallographica Section F: Structural Biology and Crystallization Communications, Vol. 62, No. 4, 01.04.2006, p. 376-380.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus

AU - Tsukazaki, Tomoya

AU - Mori, Hiroyuki

AU - Fukai, Shuya

AU - Numata, Tomoyuki

AU - Perederina, Anna

AU - Adachi, Hiroaki

AU - Matsumura, Hiroyoshi

AU - Takano, Kazufumi

AU - Murakami, Satoshi

AU - Inoue, Tsuyoshi

AU - Mori, Yusuke

AU - Sasaki, Takatomo

AU - Vassylyev, Dmitry G.

AU - Nureki, Osamu

AU - Ito, Koreaki

PY - 2006/4/1

Y1 - 2006/4/1

N2 - 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.

AB - 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.

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