+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 6h7w | ||||||||||||||||||||||||
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タイトル | Model of retromer-Vps5 complex assembled on membrane. | ||||||||||||||||||||||||
要素 |
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キーワード | PROTEIN TRANSPORT / retromer / endosome / sorting nexin / BAR / membrane trafficking | ||||||||||||||||||||||||
機能・相同性 | 機能・相同性情報 retromer, cargo-selective complex / early endosome to Golgi transport / protein retention in Golgi apparatus / retromer complex / retrograde transport, endosome to Golgi / phosphatidylinositol binding / intracellular protein transport / late endosome / endosome / endosome membrane ...retromer, cargo-selective complex / early endosome to Golgi transport / protein retention in Golgi apparatus / retromer complex / retrograde transport, endosome to Golgi / phosphatidylinositol binding / intracellular protein transport / late endosome / endosome / endosome membrane / identical protein binding / cytosol 類似検索 - 分子機能 | ||||||||||||||||||||||||
生物種 | Chaetomium thermophilum (菌類) | ||||||||||||||||||||||||
手法 | 電子顕微鏡法 / サブトモグラム平均法 / クライオ電子顕微鏡法 / 解像度: 11.4 Å | ||||||||||||||||||||||||
データ登録者 | Kovtun, O. / Leneva, N. / Ariotti, N. / Rohan, T.S. / Owen, D.J. / Briggs, J.A.G. / Collins, B.M. | ||||||||||||||||||||||||
資金援助 | 英国, オーストラリア, 7件
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引用 | ジャーナル: Nature / 年: 2018 タイトル: Structure of the membrane-assembled retromer coat determined by cryo-electron tomography. 著者: Oleksiy Kovtun / Natalya Leneva / Yury S Bykov / Nicholas Ariotti / Rohan D Teasdale / Miroslava Schaffer / Benjamin D Engel / David J Owen / John A G Briggs / Brett M Collins / 要旨: Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular ...Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions. | ||||||||||||||||||||||||
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 | 6h7w.cif.gz | 1.9 MB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb6h7w.ent.gz | 1.6 MB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 6h7w.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
文書・要旨 | 6h7w_validation.pdf.gz | 992.3 KB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 6h7w_full_validation.pdf.gz | 1.5 MB | 表示 | |
XML形式データ | 6h7w_validation.xml.gz | 219.2 KB | 表示 | |
CIF形式データ | 6h7w_validation.cif.gz | 311.5 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/h7/6h7w ftp://data.pdbj.org/pub/pdb/validation_reports/h7/6h7w | HTTPS FTP |
-関連構造データ
関連構造データ | 0154MC 0155C 0156C 0157C 0158C 0159C 0160C 0161C 0162C 0163C 5w8mC 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 |
-リンク
-集合体
登録構造単位 |
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-要素
-Vacuolar protein sorting-associated protein ... , 3種, 6分子 JCSTQR
#1: タンパク質 | 分子量: 34308.449 Da / 分子数: 2 / 由来タイプ: 組換発現 由来: (組換発現) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (菌類) 株: DSM 1495 / CBS 144.50 / IMI 039719 / 遺伝子: CTHT_0009750 / 発現宿主: Escherichia coli (大腸菌) / 参照: UniProt: G0S0E6 #5: タンパク質 | 分子量: 21470.654 Da / 分子数: 2 / 由来タイプ: 組換発現 由来: (組換発現) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (菌類) 株: DSM 1495 / CBS 144.50 / IMI 039719 / 遺伝子: CTHT_0002370 / 発現宿主: Escherichia coli (大腸菌) / 参照: UniProt: G0RZB5 #6: タンパク質 | 分子量: 96140.016 Da / 分子数: 2 / 由来タイプ: 組換発現 由来: (組換発現) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (菌類) 株: DSM 1495 / CBS 144.50 / IMI 039719 / 遺伝子: CTHT_0035730 / 発現宿主: Escherichia coli (大腸菌) / 参照: UniProt: G0S709 |
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-Putative vacuolar protein sorting-associated ... , 3種, 14分子 BAGEPNHFDKVLOM
#2: タンパク質 | 分子量: 42060.762 Da / 分子数: 8 / 由来タイプ: 組換発現 由来: (組換発現) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (菌類) 株: DSM 1495 / CBS 144.50 / IMI 039719 / 遺伝子: CTHT_0068290 / 発現宿主: Escherichia coli (大腸菌) / 参照: UniProt: G0SH11 #3: タンパク質 | 分子量: 14897.982 Da / 分子数: 4 / 由来タイプ: 組換発現 由来: (組換発現) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (菌類) 株: DSM 1495 / CBS 144.50 / IMI 039719 / 遺伝子: CTHT_0068290 / 発現宿主: Escherichia coli (大腸菌) / 参照: UniProt: G0SH11 #4: タンパク質 | 分子量: 25477.904 Da / 分子数: 2 / 由来タイプ: 組換発現 由来: (組換発現) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (菌類) 株: DSM 1495 / CBS 144.50 / IMI 039719 / 遺伝子: CTHT_0068290 / 発現宿主: Escherichia coli (大腸菌) / 参照: UniProt: G0SH11 |
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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: 3D ARRAY / 3次元再構成法: サブトモグラム平均法 |
-試料調製
構成要素 | 名称: Retromer-Vps5 complex assembled on the membrane. / タイプ: COMPLEX 詳細: Vps26/Vps35/Vps29 trimer recruited to the membrane via Vps5 PX-BAR protein. Entity ID: all / 由来: RECOMBINANT | |||||||||||||||
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分子量 | 単位: KILODALTONS/NANOMETER / 実験値: NO | |||||||||||||||
由来(天然) | 生物種: Chaetomium thermophilum var. thermophilum DSM 1495 (菌類) | |||||||||||||||
由来(組換発現) | 生物種: Escherichia coli (大腸菌) | |||||||||||||||
緩衝液 | pH: 7.5 | |||||||||||||||
緩衝液成分 |
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試料 | 濃度: 1.1 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: The solution-assembled complex was incubated with Folch liposomes at room temperature. | |||||||||||||||
急速凍結 | 装置: LEICA EM GP / 凍結剤: ETHANE / 湿度: 95 % / 凍結前の試料温度: 292 K |
-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 FIELD / 倍率(公称値): 105000 X / 最大 デフォーカス(公称値): 6500 nm / 最小 デフォーカス(公称値): 2500 nm / Cs: 2.7 mm / C2レンズ絞り径: 70 µm / アライメント法: ZEMLIN TABLEAU |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
撮影 | 電子線照射量: 3.17 e/Å2 / 検出モード: SUPER-RESOLUTION フィルム・検出器のモデル: GATAN K2 QUANTUM (4k x 4k) 撮影したグリッド数: 2 詳細: Tomographic tilt series were acquired with the dose-symmetric tilt-scheme (Hagen et al., J Struct Biol. 2017) |
電子光学装置 | エネルギーフィルター名称: GIF Quantum LS / エネルギーフィルタースリット幅: 20 eV |
画像スキャン | 動画フレーム数/画像: 10 |
-解析
EMソフトウェア |
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画像処理 | 詳細: After motion correction in "alignframes" (IMOD), each of the images in the tilt series was low-pass filtered according to the electron-dose acquired by the sample (Grant and Grigorieff, 2015). | ||||||||||||||||||||||||||||||||
CTF補正 | 詳細: CTF correction was performed with ctfphaseflip IMOD command using defocus values measured by CTFFIND4 on non-dose-filtered images. タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||
対称性 | 点対称性: C2 (2回回転対称) | ||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 11.4 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 16037 / アルゴリズム: BACK PROJECTION / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||
EM volume selection | 手法: geometrical seeding alogn the surface of manually traced tubules Num. of tomograms: 71 / Num. of volumes extracted: 194885 / Reference model: reference-free | ||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT 詳細: Note that the associated PDB model was not generated by fitting into this map! It was generated by fitting into the associated local high-resolution maps and then combined. Initial global ...詳細: Note that the associated PDB model was not generated by fitting into this map! It was generated by fitting into the associated local high-resolution maps and then combined. Initial global ridgid body fitting was done using Chimera, followed by MDFF within NAMD for flexible fitting. Note that side chain positions are not reliable in model generated by refinement into a map at this resolution. |