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基本情報
登録情報 | データベース: PDB / ID: 6u3e | ||||||
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タイトル | Best fitting antiparallel model for Volume 1 of truncated dimeric Cytohesin-3 (Grp1; amino acids 14-399) | ||||||
![]() | Cytohesin-3 | ||||||
![]() | ENDOCYTOSIS / Arf GEF / phosphoinositide binding / Sec7 domain / PH domain | ||||||
機能・相同性 | ![]() Intra-Golgi traffic / Golgi vesicle transport / establishment of epithelial cell polarity / regulation of ARF protein signal transduction / phosphatidylinositol-3,4,5-trisphosphate binding / positive regulation of cell adhesion / bicellular tight junction / ruffle / guanyl-nucleotide exchange factor activity / adherens junction ...Intra-Golgi traffic / Golgi vesicle transport / establishment of epithelial cell polarity / regulation of ARF protein signal transduction / phosphatidylinositol-3,4,5-trisphosphate binding / positive regulation of cell adhesion / bicellular tight junction / ruffle / guanyl-nucleotide exchange factor activity / adherens junction / Golgi membrane / nucleoplasm / plasma membrane / cytosol 類似検索 - 分子機能 | ||||||
生物種 | ![]() | ||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / ネガティブ染色法 / 解像度: 53 Å | ||||||
![]() | Das, S. / Lambright, D.G. | ||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes. 著者: Sanchaita Das / Andrew W Malaby / Agata Nawrotek / Wenhua Zhang / Mahel Zeghouf / Sarah Maslen / Mark Skehel / Srinivas Chakravarthy / Thomas C Irving / Osman Bilsel / Jacqueline Cherfils / David G Lambright / ![]() ![]() ![]() 要旨: Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through ...Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed. | ||||||
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構造ビューア | 分子: ![]() ![]() |
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-検証レポート
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-関連構造データ
関連構造データ | ![]() 20628MC ![]() 6u3gC M: このデータのモデリングに利用したマップデータ C: 同じ文献を引用 ( |
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集合体
登録構造単位 | ![]()
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要素
#1: タンパク質 | 分子量: 46501.766 Da / 分子数: 2 / 断片: UNP residues 13-400 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() #2: 化合物 | 研究の焦点であるリガンドがあるか | N | |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 | 名称: Truncated homodimer of Cytohesin-3 (Grp1, amino acids 14-399) with Inositol 1,3,4,5-tetrakis phosphate (IP4) タイプ: COMPLEX / Entity ID: #1 / 由来: RECOMBINANT | |||||||||||||||
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分子量 | 実験値: NO | |||||||||||||||
由来(天然) | 生物種: ![]() | |||||||||||||||
由来(組換発現) | 生物種: ![]() ![]() | |||||||||||||||
緩衝液 | pH: 8 詳細: Peak fractions after gel filtration were immediately diluted, applied to freshly glow discharged grids, and stained with uranyl formate. | |||||||||||||||
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試料 | 包埋: NO / シャドウイング: NO / 染色: YES / 凍結: NO 詳細: The sample is a uniform homodimer with significant conformational flexibility. | |||||||||||||||
染色 | タイプ: NEGATIVE / 詳細: Negatively stained with 0.75% (w/v) uranyl formate / 染色剤: Uranyl Formate | |||||||||||||||
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 400 divisions/in. |
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電子顕微鏡撮影
顕微鏡 | モデル: FEI/PHILIPS CM120T 詳細: specimen holder: FISCHIONE INSTRUMENTS DUAL AXIS TOMOGRAPHY HOLDER |
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電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 28000 X / 最大 デフォーカス(公称値): -3200 nm / 最小 デフォーカス(公称値): -1200 nm |
撮影 | 電子線照射量: 30 e/Å2 フィルム・検出器のモデル: TVIPS TEMCAM-F224 (2k x 2k) 撮影したグリッド数: 1 / 実像数: 500 |
画像スキャン | 横: 2048 / 縦: 2048 |
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解析
EMソフトウェア |
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画像処理 | 詳細: Images were processed with EMAN2 after X-ray removal with IMOD | |||||||||||||||||||||||||||||||||||
CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 6504 / 詳細: Particles were manually picked. | |||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C1 (非対称) | |||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 53 Å / 解像度の算出法: FSC 0.5 CUT-OFF / 粒子像の数: 2052 / アルゴリズム: BACK PROJECTION / クラス平均像の数: 20 / 対称性のタイプ: POINT | |||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: RIGID BODY FIT / 空間: REAL / Target criteria: Correlation coefficient 詳細: The model with the best correlation coefficient was selected by ADP_EM from a pool of 10000 models generated by RRT_SAMPLE using rigid bodies derived from 2R09 (autoinhibited core, amino ...詳細: The model with the best correlation coefficient was selected by ADP_EM from a pool of 10000 models generated by RRT_SAMPLE using rigid bodies derived from 2R09 (autoinhibited core, amino acids 63-399) and a canonical antiparallel coiled coil (amino acids 18-53) built with CCBuilder. Flexible regions with reasonable geometry were modeled with MODELLER. | |||||||||||||||||||||||||||||||||||
原子モデル構築 | 3D fitting-ID: 1 / Accession code: 2R09 / Initial refinement model-ID: 1 / Pdb chain residue range: 63-399 / PDB-ID: 2R09 / Source name: PDB / タイプ: experimental model
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