- PDB-3iz2: C-alpha model fitted into the EM structure of Cx26M34Adel2-7 -
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基本情報
登録情報
データベース: PDB / ID: 3iz2
タイトル
C-alpha model fitted into the EM structure of Cx26M34Adel2-7
要素
Gap junction beta-2 protein
キーワード
MEMBRANE PROTEIN / Gap Junction channel
機能・相同性
機能・相同性情報
Transport of connexons to the plasma membrane / response to human chorionic gonadotropin / gap junction-mediated intercellular transport / gap junction channel activity involved in cell communication by electrical coupling / epididymis development / Oligomerization of connexins into connexons / Transport of connexins along the secretory pathway / gap junction assembly / connexin complex / gap junction ...Transport of connexons to the plasma membrane / response to human chorionic gonadotropin / gap junction-mediated intercellular transport / gap junction channel activity involved in cell communication by electrical coupling / epididymis development / Oligomerization of connexins into connexons / Transport of connexins along the secretory pathway / gap junction assembly / connexin complex / gap junction / gap junction channel activity / astrocyte projection / Gap junction assembly / endoplasmic reticulum-Golgi intermediate compartment / inner ear development / decidualization / lateral plasma membrane / response to retinoic acid / cellular response to glucagon stimulus / cellular response to dexamethasone stimulus / response to progesterone / response to ischemia / sensory perception of sound / transmembrane transport / cell-cell signaling / response to estradiol / cell body / cellular response to oxidative stress / response to lipopolysaccharide / calcium ion binding / perinuclear region of cytoplasm / identical protein binding / plasma membrane 類似検索 - 分子機能
Gap junction beta-2 protein (Cx26) / Connexin / Connexin, N-terminal / Connexin, conserved site / Gap junction protein, cysteine-rich domain / Connexin, N-terminal domain superfamily / Connexin / Connexins signature 1. / Connexins signature 2. / Connexin homologues / Gap junction channel protein cysteine-rich domain 類似検索 - ドメイン・相同性
ジャーナル: J Mol Biol / 年: 2011 タイトル: Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels. 著者: Atsunori Oshima / Kazutoshi Tani / Masoud M Toloue / Yoko Hiroaki / Amy Smock / Sayaka Inukai / Angela Cone / Bruce J Nicholson / Gina E Sosinsky / Yoshinori Fujiyoshi / 要旨: Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we ...Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we present electron crystallographic structures of a mutant human connexin26 (Cx26M34A) and an N-terminal deletion of this mutant (Cx26M34Adel2-7) at 6-Å and 10-Å resolutions, respectively. The three-dimensional map of Cx26M34A was improved by data from 60° tilt images and revealed a breakdown of the hexagonal symmetry in a connexin hemichannel, particularly in the cytoplasmic domain regions at the ends of the transmembrane helices. The Cx26M34A structure contained an asymmetric density in the channel vestibule ("plug") that was decreased in the Cx26M34Adel2-7 structure, indicating that the N terminus significantly contributes to form this plug feature. Functional analysis of the Cx26M34A channels revealed that these channels are predominantly closed, with the residual electrical conductance showing normal voltage gating. N-terminal deletion mutants with and without the M34A mutation showed no electrical activity in paired Xenopus oocytes and significantly decreased dye permeability in HeLa cells. Comparing this closed structure with the recently published X-ray structure of wild-type Cx26, which is proposed to be in an open state, revealed a radial outward shift in the transmembrane helices in the closed state, presumably to accommodate the N-terminal plug occluding the pore. Because both Cx26del2-7 and Cx26M34Adel2-7 channels are closed, the N terminus appears to have a prominent role in stabilizing the open configuration.
名称: connexin26 gap junction channels / タイプ: COMPLEX
緩衝液
名称: 10mM MES / pH: 5.8 詳細: 10% trehalose (Sigma, St. Louis, MO), 10 mM MES (pH 5.8), 300 mM NaCl, 50 mM MgCl2, 5 mM CaCl2, 2 mM dithiothreitol, 100 microM carbenoxolone, 0.005% NaN3, and 1% glycerol10mM MES
試料
濃度: 2 mg/ml / 包埋: YES / シャドウイング: NO / 染色: NO / 凍結: YES
試料支持
グリッドの材料: MOLYBDENUM
EM embedding
詳細: 10% trehalose / Material: trehalose
急速凍結
装置: REICHERT-JUNG PLUNGER / 凍結剤: NITROGEN / Temp: 93 K 手法: The grids were blotted with filter paper and fast frozen into liquid nitrogen
モード: BRIGHT FIELD / 倍率(公称値): 40000 X / 倍率(補正後): 39000 X / 最大 デフォーカス(公称値): 2214 nm / 最小 デフォーカス(公称値): 544 nm / Cs: 1.6 mm
試料ホルダ
温度: 4.2 K / 傾斜角・最大: 45 ° / 傾斜角・最小: 0 °
撮影
電子線照射量: 25 e/Å2 / フィルム・検出器のモデル: KODAK SO-163 FILM
放射
プロトコル: SINGLE WAVELENGTH / 単色(M)・ラウエ(L): M / 散乱光タイプ: electron
放射波長
相対比: 1
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解析
EMソフトウェア
ID
名称
カテゴリ
1
Situs
モデルフィッティング
2
MRC
3次元再構成
CTF補正
詳細: Each image
3次元再構成
手法: Lattice line fitting / 解像度: 10 Å / 倍率補正: carbon grating / 対称性のタイプ: 2D CRYSTAL
原子モデル構築
プロトコル: RIGID BODY FIT / 空間: REAL / Target criteria: Cross-correlation coefficient 詳細: REFINEMENT PROTOCOL--Rigid body DETAILS--Initial local fitting was done using O, and Situs was used for flexible fitting.