+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 7ndg | |||||||||||||||||||||||||||||||||
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タイトル | Cryo-EM structure of the ternary complex between Netrin-1, Neogenin and Repulsive Guidance Molecule B | |||||||||||||||||||||||||||||||||
要素 |
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キーワード | SIGNALING PROTEIN / Signal transduction (シグナル伝達) / cell surface receptors / neuron regeneration / cell migration (遊走) / Netrin / Neogenin / Repulsive Guidance Molecule / complex structure / protein-protein interactions (タンパク質間相互作用) | |||||||||||||||||||||||||||||||||
機能・相同性 | 機能・相同性情報 regulation of glial cell migration / chemorepulsion of axon / anterior/posterior axon guidance / Cdc42 protein signal transduction / Role of second messengers in netrin-1 signaling / Netrin-1 signaling / motor neuron migration / Regulation of commissural axon pathfinding by SLIT and ROBO / negative regulation of axon extension / Netrin mediated repulsion signals ...regulation of glial cell migration / chemorepulsion of axon / anterior/posterior axon guidance / Cdc42 protein signal transduction / Role of second messengers in netrin-1 signaling / Netrin-1 signaling / motor neuron migration / Regulation of commissural axon pathfinding by SLIT and ROBO / negative regulation of axon extension / Netrin mediated repulsion signals / substrate-dependent cell migration, cell extension / mammary gland duct morphogenesis / positive regulation of cell motility / nuclear migration / DCC mediated attractive signaling / regulation of synapse assembly / inner ear morphogenesis / DSCAM interactions / 基底膜 / endoplasmic reticulum-Golgi intermediate compartment / positive regulation of axon extension / glial cell proliferation / BMP signaling pathway / coreceptor activity / side of membrane / positive regulation of glial cell proliferation / 細胞接着 / マイクロフィラメント / Ras protein signal transduction / DNA-binding transcription factor activity, RNA polymerase II-specific / 細胞接着 / 脂質ラフト / RNA polymerase II cis-regulatory region sequence-specific DNA binding / apoptotic process / regulation of transcription by RNA polymerase II / positive regulation of DNA-templated transcription / シグナル伝達 / extracellular region / 核質 / 生体膜 / identical protein binding / 細胞膜 / 細胞質基質 類似検索 - 分子機能 | |||||||||||||||||||||||||||||||||
生物種 | Homo sapiens (ヒト) Mus musculus (ハツカネズミ) | |||||||||||||||||||||||||||||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 5.98 Å | |||||||||||||||||||||||||||||||||
データ登録者 | Robinson, R.A. / Griffiths, S.C. / van de Haar, L.L. / Malinauskas, T. / van Battum, E.Y. / Zelina, P. / Schwab, R.A. / Karia, D. / Malinauskaite, L. / Brignani, S. ...Robinson, R.A. / Griffiths, S.C. / van de Haar, L.L. / Malinauskas, T. / van Battum, E.Y. / Zelina, P. / Schwab, R.A. / Karia, D. / Malinauskaite, L. / Brignani, S. / van den Munkhof, M. / Dudukcu, O. / De Ruiter, A.A. / Van den Heuvel, D.M.A. / Bishop, B. / Elegheert, J. / Aricescu, A.R. / Pasterkamp, R.J. / Siebold, C. | |||||||||||||||||||||||||||||||||
資金援助 | 英国, オランダ, 10件
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引用 | ジャーナル: Cell / 年: 2021 タイトル: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling. 著者: Ross A Robinson / Samuel C Griffiths / Lieke L van de Haar / Tomas Malinauskas / Eljo Y van Battum / Pavol Zelina / Rebekka A Schwab / Dimple Karia / Lina Malinauskaite / Sara Brignani / ...著者: Ross A Robinson / Samuel C Griffiths / Lieke L van de Haar / Tomas Malinauskas / Eljo Y van Battum / Pavol Zelina / Rebekka A Schwab / Dimple Karia / Lina Malinauskaite / Sara Brignani / Marleen H van den Munkhof / Özge Düdükcü / Anna A De Ruiter / Dianne M A Van den Heuvel / Benjamin Bishop / Jonathan Elegheert / A Radu Aricescu / R Jeroen Pasterkamp / Christian Siebold / 要旨: During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin ...During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs. | |||||||||||||||||||||||||||||||||
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 | 分子: MolmilJmol/JSmol |
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 7ndg.cif.gz | 537.2 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb7ndg.ent.gz | 437 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 7ndg.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/nd/7ndg ftp://data.pdbj.org/pub/pdb/validation_reports/nd/7ndg | HTTPS FTP |
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-関連構造データ
関連構造データ | 12286MC 7ne0C 7ne1C 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 (文献) |
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類似構造データ | |
電子顕微鏡画像生データ | EMPIAR-10637 (タイトル: Cryo-EM structure of the ternary Netrin 1-Neogenin 1-Repulsive Guidance Molecule B complex Data size: 1.2 TB Data #1: Unaligned multi-frame micrographs of the ternary Netrin 1-Neogenin 1-Repulsive Guidance Molecule B complex [micrographs - multiframe]) |
-リンク
-集合体
登録構造単位 |
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1 |
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-要素
-タンパク質 , 4種, 12分子 AGDBHEFCINMO
#1: タンパク質 | 分子量: 49227.402 Da / 分子数: 3 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: NTN1, NTN1L / プラスミド: pHLsec 詳細 (発現宿主): Aricescu et al., Acta Crystallogr D Biol Crystallogr, 2006, PMID 17001101 細胞株 (発現宿主): HEK293T / 器官 (発現宿主): Kidney / 発現宿主: Homo sapiens (ヒト) / 参照: UniProt: O95631 #2: タンパク質 | 分子量: 39268.199 Da / 分子数: 3 / 由来タイプ: 組換発現 / 由来: (組換発現) Mus musculus (ハツカネズミ) / 遺伝子: Neo1 / プラスミド: pHLsec 詳細 (発現宿主): Aricescu et al., Acta Crystallogr D Biol Crystallogr, 2006, PMID 17001101 細胞株 (発現宿主): HEK293T / 器官 (発現宿主): Kidney / 発現宿主: Homo sapiens (ヒト) / 参照: UniProt: Q7TQG5 #3: タンパク質 | 分子量: 28104.494 Da / 分子数: 3 / 由来タイプ: 組換発現 / 詳細: Repulsive Guidance Molecule B / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: RGMB / 細胞株 (発現宿主): HEK293T / 発現宿主: Homo sapiens (ヒト) / 参照: UniProt: Q6NW40 #4: タンパク質 | 分子量: 13022.377 Da / 分子数: 3 / 由来タイプ: 組換発現 / 詳細: Repulsive Guidance Molecule B / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: RGMB / 細胞株 (発現宿主): HEK293T / 発現宿主: Homo sapiens (ヒト) / 参照: UniProt: Q6NW40 |
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-非ポリマー / 糖 , 2種, 15分子
#5: 化合物 | #6: 糖 | ChemComp-NAG / |
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-詳細
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 |
-試料調製
構成要素 |
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分子量 |
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由来(天然) |
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由来(組換発現) |
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緩衝液 | pH: 7.5 詳細: 10 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl2, 1 mM sucrose octasulfate, 0.01% NaN3 | ||||||||||||||||||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.07 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: The ternary NEO1-NET1-RGMB complex was purified by SEC on a S200 10/300 Increase column with a running buffer of 10 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl2, 1 mM sucrose octasulfate, 0.01% ...詳細: The ternary NEO1-NET1-RGMB complex was purified by SEC on a S200 10/300 Increase column with a running buffer of 10 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl2, 1 mM sucrose octasulfate, 0.01% NaN3 at 4 degrees C . The peak fraction containing the ternary complex was diluted to 0.07 mg per ml in SEC buffer. | ||||||||||||||||||||||||||||||||||||
試料支持 | 詳細: Agar Scientific Ultra-thin carbon support film, 3 nm - on lacey carbon. https://www.agarscientific.com/ultra-thin-carbon-support-film-3-nm-on-holey-carbon グリッドの材料: COPPER グリッドのタイプ: PELCO Ultrathin Carbon with Lacey Carbon | ||||||||||||||||||||||||||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 95 % / 凍結前の試料温度: 277.15 K 詳細: Lacey carbon grids with 3 nm ultrathin carbon support film were glow discharged for 30 seconds at high RF level using Harrick Plasma Cleaner, model PDC-002-CE, and then 3.5 microl of the ...詳細: Lacey carbon grids with 3 nm ultrathin carbon support film were glow discharged for 30 seconds at high RF level using Harrick Plasma Cleaner, model PDC-002-CE, and then 3.5 microl of the sample was pipetted per grid. Excess protein was blotted away for 3 seconds using filter paper (round filter paper for Vitrobot from Agar Scientific, catalogue number 47000-100) and Vitrobot Mark IV (Thermo Fisher Scientific) (relative force -15) at 95-100% humidity. Grids were plunge frozen in liquid ethane. |
-電子顕微鏡撮影
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELDBright-field microscopy / 倍率(公称値): 96000 X / 最大 デフォーカス(公称値): -700 nm / 最小 デフォーカス(公称値): -500 nm / C2レンズ絞り径: 50 µm / アライメント法: BASIC |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER 最高温度: 77 K / 最低温度: 77 K |
撮影 | 平均露光時間: 37.26 sec. / 電子線照射量: 40 e/Å2 / 検出モード: COUNTING フィルム・検出器のモデル: FEI FALCON III (4k x 4k) 撮影したグリッド数: 1 / 実像数: 1635 詳細: Cryo-EM data were collected on a Titan Krios G3i microscope (Thermo Fisher Scientific) operating at 300 kV with a 50 microm C2 aperture and Volta phase plate (Thermo Fisher Scientific), at ...詳細: Cryo-EM data were collected on a Titan Krios G3i microscope (Thermo Fisher Scientific) operating at 300 kV with a 50 microm C2 aperture and Volta phase plate (Thermo Fisher Scientific), at the Division of Structural Biology, University of Oxford. Movies were recorded using a FEI Falcon III direct electron detector in electron counting mode using EPU software at a nominal magnification of 96000x, corresponding to a physical pixel size of 0.85 angstrom/pixel. A total dose of 40 electrons per square angstrom was used at a dose rate of 0.77 electrons/pix/sec. |
電子光学装置 | 位相板: VOLTA PHASE PLATE |
-解析
EMソフトウェア |
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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: 280158 | ||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C3 (3回回転対称) | ||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 5.98 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 68541 詳細: In total 1635 movies were collected and drift correction, beam-induced motion and dose-weighting were performed with MotionCor2 RELION 3.1 (Zivanov et al., 2018) for 1635 movies. Contrast ...詳細: In total 1635 movies were collected and drift correction, beam-induced motion and dose-weighting were performed with MotionCor2 RELION 3.1 (Zivanov et al., 2018) for 1635 movies. Contrast transfer function (CTF) was estimated using CTFFIND 4.1 (Rohou and Grigorieff, 2015) implemented in RELION. 280158 particles were picked using Warp (Tegunov and Cramer, 2019). 2D classification in cryoSPARC v2 (Punjani et al., 2017) were performed for these particles and best 2D class averaged with 100674 particles were used to generate ab-initio 3D model with C3 symmetry. C1 symmetry did not generate reasonable 3D model. All Warp picked particles were used for 3D classification in cryoSPARC v2 and the best class with 177056 particles was used for refinement in RELION 3.1 with initial model generated in cryoSPARC v2. Byesian particle polishing improved the resolution to 5.44 angstrom, however the map for RGMB was very weak. Last step of 3D classification without alignment with T regularisation parameter set to 16 was performed and gave one class with more continuous map for RGMB, which was refined to 5.98 angstrom resolution, as estimated using the Fourier shell correlation (FSC) = 0.143 criterion. クラス平均像の数: 1 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: RIGID BODY FIT / Target criteria: Correlation coefficient 詳細: Crystal structure used as a model for fitting and rigid body refinement will be described by Robinson, Griffiths, van de Haar, Malinauskas et al., Cell, 2021. |