+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-1766 | |||||||||
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タイトル | Single particle analysis of a tetramer of a subunit comprising the 1:1 complex of PSD-95 and Kir2.1NC_4, in negative stain | |||||||||
マップデータ | Negative stain EM tetrad (C_4 symmetry applied) of a unit comprising PSD-95 and the Kir2.1NC_4 homotetramer | |||||||||
試料 |
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キーワード | Clustering / SCAFFOLD PROTEIN / MEMBRANE ASSOCIATED GUANYLATEKINASE / PDZ / SH3 / Ion Channel / cytoplasmic domain / inwardly rectifying / membrane protein / homotetramer | |||||||||
機能・相同性 | Potassium channel, inwardly rectifying, Kir2.1 / neuronal ion channel clustering / Disks large 1-like / membrane => GO:0016020 機能・相同性情報 | |||||||||
生物種 | Mus musculus (ハツカネズミ) / Rattus norvegicus (ドブネズミ) | |||||||||
手法 | 単粒子再構成法 / ネガティブ染色法 / 解像度: 25.8 Å | |||||||||
データ登録者 | Fomina S / Howard TD / Sleator OK / Golovanova M / O'Ryan L / Leyland M / Grossmann JG / Collins RF / Prince SM | |||||||||
引用 | ジャーナル: Biochim Biophys Acta / 年: 2011 タイトル: Self-directed assembly and clustering of the cytoplasmic domains of inwardly rectifying Kir2.1 potassium channels on association with PSD-95. 著者: Svetlana Fomina / Tina D Howard / Olivia K Sleator / Marina Golovanova / Liam O'Ryan / Mark L Leyland / J Günter Grossmann / Richard F Collins / Stephen M Prince / 要旨: The interaction of the extra-membranous domain of tetrameric inwardly rectifying Kir2.1 ion channels (Kir2.1NC(4)) with the membrane associated guanylate kinase protein PSD-95 has been studied using ...The interaction of the extra-membranous domain of tetrameric inwardly rectifying Kir2.1 ion channels (Kir2.1NC(4)) with the membrane associated guanylate kinase protein PSD-95 has been studied using Transmission Electron Microscopy in negative stain. Three types of complexes were observed in electron micrographs corresponding to a 1:1 complex, a large self-enclosed tetrad complex and extended chains of linked channel domains. Using models derived from small angle X-ray scattering experiments in which high resolution structures from X-ray crystallographic and Nuclear Magnetic Resonance studies are positioned, the envelopes from single particle analysis can be resolved as a Kir2.1NC(4):PSD-95 complex and a tetrad of this unit (Kir2.1NC(4):PSD-95)(4). The tetrad complex shows the close association of the Kir2.1 cytoplasmic domains and the influence of PSD-95 mediated self-assembly on the clustering of these channels. | |||||||||
履歴 |
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-構造の表示
ムービー |
ムービービューア |
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構造ビューア | EMマップ: SurfViewMolmilJmol/JSmol |
添付画像 |
-ダウンロードとリンク
-EMDBアーカイブ
マップデータ | emd_1766.map.gz | 972.7 KB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-1766-v30.xml emd-1766.xml | 13.8 KB 13.8 KB | 表示 表示 | EMDBヘッダ |
画像 | emd_1766.png | 161.2 KB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-1766 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1766 | HTTPS FTP |
-関連構造データ
-リンク
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
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-マップ
ファイル | ダウンロード / ファイル: emd_1766.map.gz / 形式: CCP4 / 大きさ: 1.9 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | Negative stain EM tetrad (C_4 symmetry applied) of a unit comprising PSD-95 and the Kir2.1NC_4 homotetramer | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 3.667 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
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-添付データ
-試料の構成要素
-全体 : Complex of mouse Kir2.1, cytoplasmic domain, homotetramer of fuse...
全体 | 名称: Complex of mouse Kir2.1, cytoplasmic domain, homotetramer of fused N,C termini with rat PSD-95 |
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要素 |
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-超分子 #1000: Complex of mouse Kir2.1, cytoplasmic domain, homotetramer of fuse...
超分子 | 名称: Complex of mouse Kir2.1, cytoplasmic domain, homotetramer of fused N,C termini with rat PSD-95 タイプ: sample / ID: 1000 詳細: Sample prepared by immobilizing PSD-95 on an affinity column followed by incubation with purified Kir2.1NC homotetramers. 集合状態: One tetramer of Kir2.1NC binds to PSD-95 / Number unique components: 2 |
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分子量 | 理論値: 872 KDa / 手法: Sum of component MW |
-分子 #1: Kir2.1 cytoplasmic domain
分子 | 名称: Kir2.1 cytoplasmic domain / タイプ: protein_or_peptide / ID: 1 / Name.synonym: Kir2.1NC / コピー数: 4 / 集合状態: Tetramer / 組換発現: Yes |
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由来(天然) | 生物種: Mus musculus (ハツカネズミ) / 別称: Mouse / 細胞中の位置: Membrane |
分子量 | 実験値: 140 KDa / 理論値: 140 KDa |
組換発現 | 生物種: Escherichia coli (大腸菌) / 組換プラスミド: pET15b |
配列 | GO: membrane => GO:0016020 / InterPro: Potassium channel, inwardly rectifying, Kir2.1 |
-分子 #2: PSD-95
分子 | 名称: PSD-95 / タイプ: protein_or_peptide / ID: 2 / Name.synonym: PSD-95 / コピー数: 1 / 集合状態: Monomer / 組換発現: Yes |
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由来(天然) | 生物種: Rattus norvegicus (ドブネズミ) / 別称: Norway rat |
分子量 | 実験値: 95 KDa / 理論値: 78 KDa |
組換発現 | 生物種: Escherichia coli (大腸菌) / 組換プラスミド: pGEX-6P |
配列 | GO: neuronal ion channel clustering / InterPro: Disks large 1-like |
-実験情報
-構造解析
手法 | ネガティブ染色法 |
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解析 | 単粒子再構成法 |
試料の集合状態 | particle |
-試料調製
緩衝液 | pH: 7.5 詳細: 20mM Tris/HCl, 150mM NaCl, 1mM reduced GSH, 1mM EDTA, 50mM L-Glutamic acid, 50mM L-Arginine |
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染色 | タイプ: NEGATIVE 詳細: Samples were adsorbed onto freshly glow discharged carbon film grids. Sample solution was pipetted into the grid followed by blotting, de-ionized water was then applied for 10s followed by ...詳細: Samples were adsorbed onto freshly glow discharged carbon film grids. Sample solution was pipetted into the grid followed by blotting, de-ionized water was then applied for 10s followed by blotting, 2%w/v Uranyl Acetate solution was applied followed by a final blotting step. |
グリッド | 詳細: 400 mesh Copper |
凍結 | 凍結剤: NONE / 装置: OTHER |
-電子顕微鏡法
顕微鏡 | FEI TECNAI 10 |
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詳細 | Low dose |
撮影 | カテゴリ: FILM / フィルム・検出器のモデル: GENERIC FILM / デジタル化 - スキャナー: OTHER / デジタル化 - サンプリング間隔: 15 µm / 実像数: 31 / Od range: 2 / ビット/ピクセル: 8 |
Tilt angle min | 0 |
Tilt angle max | 0 |
電子線 | 加速電圧: 100 kV / 電子線源: TUNGSTEN HAIRPIN |
電子光学系 | 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 3.6 mm / 最大 デフォーカス(公称値): 1.6 µm / 最小 デフォーカス(公称値): 0.6 µm / 倍率(公称値): 43000 |
試料ステージ | 試料ホルダー: Eucentric / 試料ホルダーモデル: PHILIPS ROTATION HOLDER |
-画像解析
詳細 | A tomographic reconstruction was initially generated, this was then used to select Particles using model based picking. |
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CTF補正 | 詳細: Parameters determined using a calculated Scattering curve |
最終 再構成 | 想定した対称性 - 点群: C4 (4回回転対称) / 解像度のタイプ: BY AUTHOR / 解像度: 25.8 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ソフトウェア - 名称: EMAN / 使用した粒子像数: 14433 |
最終 2次元分類 | クラス数: 98 |
-原子モデル構築 1
初期モデル | PDB ID: |
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ソフトウェア | 名称: Chimera |
詳細 | (tetrad) ROTATION MATRIX -0.47579 -0.11676 -0.87178 0.80871 -0.44780 -0.38139 -0.34585 -0.88647 0.30749 TRANSLATION VECTOR IN AS -29.84596 -32.38952 6.20368 subsequently apply the following to both sets of transformed coordinates Symmetry Transformation 1 ROTATION MATRIX 1.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 1.000 TRANSLATION VECTOR IN AS 0.000 0.000 0.000 Symmetry Transformation 2 ROTATION MATRIX -1.000 0.000 0.000 0.000 -1.000 0.000 0.000 0.000 1.000 TRANSLATION VECTOR IN AS 0.000 0.000 0.000 Symmetry Transformation 3 ROTATION MATRIX 0.000 -1.000 0.000 1.000 0.000 0.000 0.000 0.000 1.000 TRANSLATION VECTOR IN AS 0.000 0.000 0.000 Symmetry Transformation 4 ROTATION MATRIX 0.000 1.000 0.000 -1.000 0.000 0.000 0.000 0.000 1.000 TRANSLATION VECTOR IN AS 0.000 0.000 0.000 |
精密化 | 空間: REAL |
-原子モデル構築 2
初期モデル | PDB ID: |
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ソフトウェア | 名称: Chimera |
詳細 | Tetrad ROTATION MATRIX -0.74744 -0.64448 0.16117 0.66166 -0.74393 0.09367 0.05953 0.17665 0.98247 TRANSLATION VECTOR IN AS -28.01615 -38.28474 -19.16218 subsequently apply the following to both sets of transformed coordinates Symmetry Transformation 1 ROTATION MATRIX 1.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 1.000 TRANSLATION VECTOR IN AS 0.000 0.000 0.000 Symmetry Transformation 2 ROTATION MATRIX -1.000 0.000 0.000 0.000 -1.000 0.000 0.000 0.000 1.000 TRANSLATION VECTOR IN AS 0.000 0.000 0.000 Symmetry Transformation 3 ROTATION MATRIX 0.000 -1.000 0.000 1.000 0.000 0.000 0.000 0.000 1.000 TRANSLATION VECTOR IN AS 0.000 0.000 0.000 Symmetry Transformation 4 ROTATION MATRIX 0.000 1.000 0.000 -1.000 0.000 0.000 0.000 0.000 1.000 TRANSLATION VECTOR IN AS 0.000 0.000 0.000 |
精密化 | 空間: REAL |