A description of the structural determination procedures of a gap junction channel at 3.5 Å resolution

Michihiro Suga, Shoji Maeda, So Nakagawa, Eiki Yamashita, Tomitake Tsukihara

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Intercellular signalling is an essential characteristic of multicellular organisms. Gap junctions, which consist of arrays of intercellular channels, permit the exchange of ions and small molecules between adjacent cells. Here, the structural deter-mination of a gap junction channel composed of connexin 26 (Cx26) at 3.5 Å resolution is described. During each step of the purification process, the protein was examined using electron microscopy and/or dynamic light scattering. Dehydration of the crystals improved the resolution limits. Phase refinement using multi-crystal averaging in conjunction with noncrystallographic symmetry averaging based on strictly determined noncrystallographic symmetry operators resulted in an electron-density map for model building. The amino-acid sequence of a protomer structure consisting of the amino-terminal helix, four transmembrane helices and two extracellular loops was assigned to the electron-density map. The amino-acid assignment was confirmed using six selenomethionine (SeMet) sites in the difference Fourier map of the SeMet derivative and three intramolecular disulfide bonds in the anomalous difference Fourier map of the native crystal.

Original languageEnglish
Pages (from-to)758-766
Number of pages9
JournalActa Crystallographica Section D: Biological Crystallography
Volume65
Issue number8
DOIs
Publication statusPublished - 2009
Externally publishedYes

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Selenomethionine
Gap Junctions
Electrons
Ion Exchange
Protein Subunits
Dehydration
Disulfides
Amino Acid Sequence
Electron Microscopy
Amino Acids
Proteins
Connexin 26
Dynamic Light Scattering

Keywords

  • Gap junction channel
  • Membrane proteins
  • Multi-crystal averaging

ASJC Scopus subject areas

  • Structural Biology

Cite this

A description of the structural determination procedures of a gap junction channel at 3.5 Å resolution. / Suga, Michihiro; Maeda, Shoji; Nakagawa, So; Yamashita, Eiki; Tsukihara, Tomitake.

In: Acta Crystallographica Section D: Biological Crystallography, Vol. 65, No. 8, 2009, p. 758-766.

Research output: Contribution to journalArticle

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