A description of the structural determination procedures of a gap junction channel at 3.5 A resolution.
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 determination of a gap junction channel composed of connexin 26 (Cx26) at 3.5 A 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.
Suga
Michihiro
M
Institute for Protein Research, Osaka University, OLABB, 6-2-6 Furuedai, Suita, Osaka 565-0874, Japan.
Maeda
Shoji
S
Nakagawa
So
S
Yamashita
Eiki
E
Tsukihara
Tomitake
T
D016367:Baculoviridae; D003001:Cloning, Molecular; D000072259:Connexin 26; D017630:Connexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D003681:Dehydration; D005583:Fourier Analysis; D017629:Gap Junctions / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D008854:Microscopy, Electron; D008956:Models, Chemical; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012645:Selenomethionine / Q000737:chemistry / Q000378:metabolism
D017630:Connexins; C000607042:GJB2 protein, human; D011994:Recombinant Proteins; D000072259:Connexin 26; D012645:Selenomethionine