+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-31600 | |||||||||
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タイトル | The cryo-EM map of the core region from the X. laevis NPC CR subunit | |||||||||
マップデータ | composite map for the Xenopus laevie CR subunit. | |||||||||
試料 |
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キーワード | CR / NPC / nucleoporin / Nup358 / Nup93 / Y complex / STRUCTURAL PROTEIN | |||||||||
機能・相同性 | 機能・相同性情報 GATOR2 complex / nephron development / protein localization to nuclear inner membrane / COPII-coated vesicle budding / nuclear pore inner ring / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore outer ring / nuclear pore complex assembly / nuclear pore organization / COPII vesicle coat ...GATOR2 complex / nephron development / protein localization to nuclear inner membrane / COPII-coated vesicle budding / nuclear pore inner ring / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore outer ring / nuclear pore complex assembly / nuclear pore organization / COPII vesicle coat / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / attachment of mitotic spindle microtubules to kinetochore / structural constituent of nuclear pore / RNA export from nucleus / poly(A)+ mRNA export from nucleus / mitotic metaphase chromosome alignment / NLS-bearing protein import into nucleus / cellular response to nutrient levels / ribosomal large subunit export from nucleus / mRNA transport / ribosomal small subunit export from nucleus / mRNA export from nucleus / nuclear pore / positive regulation of TORC1 signaling / cellular response to amino acid starvation / nuclear periphery / GTPase activator activity / kinetochore / protein import into nucleus / protein transport / nuclear membrane / cell division / lysosomal membrane / structural molecule activity / positive regulation of DNA-templated transcription / metal ion binding / cytoplasm / cytosol 類似検索 - 分子機能 | |||||||||
生物種 | Xenopus laevis (アフリカツメガエル) | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.7 Å | |||||||||
データ登録者 | Shi Y / Huang G / Zhan X | |||||||||
資金援助 | 中国, 1件
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引用 | ジャーナル: Science / 年: 2022 タイトル: Structure of the cytoplasmic ring of the nuclear pore complex. 著者: Xuechen Zhu / Gaoxingyu Huang / Chao Zeng / Xiechao Zhan / Ke Liang / Qikui Xu / Yanyu Zhao / Pan Wang / Qifan Wang / Qiang Zhou / Qinghua Tao / Minhao Liu / Jianlin Lei / Chuangye Yan / Yigong Shi / 要旨: INTRODUCTION The nuclear pore complex (NPC) resides on the nuclear envelope (NE) and mediates nucleocytoplasmic cargo transport. As one of the largest cellular machineries, a vertebrate NPC consists ...INTRODUCTION The nuclear pore complex (NPC) resides on the nuclear envelope (NE) and mediates nucleocytoplasmic cargo transport. As one of the largest cellular machineries, a vertebrate NPC consists of cytoplasmic filaments, a cytoplasmic ring (CR), an inner ring, a nuclear ring, a nuclear basket, and a luminal ring. Each NPC has eight repeating subunits. Structure determination of NPC is a prerequisite for understanding its functional mechanism. In the past two decades, integrative modeling, which combines x-ray structures of individual nucleoporins and subcomplexes with cryo-electron tomography reconstructions, has played a crucial role in advancing our knowledge about the NPC. The CR has been a major focus of structural investigation. The CR subunit of human NPC was reconstructed by cryo-electron tomography through subtomogram averaging to an overall resolution of ~20 Å, with local resolution up to ~15 Å. Each CR subunit comprises two Y-shaped multicomponent complexes known as the inner and outer Y complexes. Eight inner and eight outer Y complexes assemble in a head-to-tail fashion to form the proximal and distal rings, respectively, constituting the CR scaffold. To achieve higher resolution of the CR, we used single-particle cryo-electron microscopy (cryo-EM) to image the intact NPC from the NE of oocytes. Reconstructions of the core region and the Nup358 region of the CR subunit had been achieved at average resolutions of 5 to 8 Å, allowing identification of secondary structural elements. RATIONALE Packing interactions among the components of the CR subunit were poorly defined by all previous EM maps. Additional components of the CR subunit are strongly suggested by the EM maps of 5- to 8-Å resolution but remain to be identified. Addressing these issues requires improved resolution of the cryo-EM reconstruction. Therefore, we may need to enhance sample preparation, optimize image acquisition, and develop an effective data-processing strategy. RESULTS To reduce conformational heterogeneity of the sample, we spread the opened NE onto the grids with minimal force and used the chemical cross-linker glutaraldehyde to stabilize the NPC. To alleviate orientation bias of the NPC, we tilted sample grids and imaged the sample with higher electron dose at higher angles. We improved the image-processing protocol. With these efforts, the average resolutions for the core and the Nup358 regions have been improved to 3.7 and 4.7 Å, respectively. The highest local resolution of the core region reaches 3.3 Å. In addition, a cryo-EM structure of the N-terminal α-helical domain of Nup358 has been resolved at 3.0-Å resolution. These EM maps allow the identification of five copies of Nup358, two copies of Nup93, two copies of Nup205, and two copies of Y complexes in each CR subunit. Relying on the EM maps and facilitated by AlphaFold prediction, we have generated a final model for the CR of the NPC. Our model of the CR subunit includes 19,037 amino acids in 30 nucleoporins. A previously unknown C-terminal fragment of Nup160 was found to constitute a key part of the vertex, in which the short arm, long arm, and stem of the Y complex meet. The Nup160 C-terminal fragment directly binds the β-propeller proteins Seh1 and Sec13. Two Nup205 molecules, which do not contact each other, bind the inner and outer Y complexes through distinct interfaces. Conformational elasticity of the two Nup205 molecules may underlie their versatility in binding to different nucleoporins in the proximal and distal CR rings. Two Nup93 molecules, each comprising an N-terminal extended helix and an ACE1 domain, bridge the Y complexes and Nup205. Nup93 and Nup205 together play a critical role in mediating the contacts between neighboring CR subunits. Five Nup358 molecules, each in the shape of a shrimp tail and named "the clamp," hold the stems of both Y complexes. The innate conformational elasticity allows each Nup358 clamp to adapt to a distinct local environment for optimal interactions with neighboring nucleoporins. In each CR subunit, the α-helical nucleoporins appear to provide the conformational elasticity; the 12 β-propellers may strengthen the scaffold. CONCLUSION Our EM map-based model of the CR subunit substantially expands the molecular mass over the reported composite models of vertebrate CR subunit. In addition to the Y complexes, five Nup358, two Nup205, and two Nup93 molecules constitute the key components of the CR. The improved EM maps reveal insights into the interfaces among the nucleoporins of the CR. [Figure: see text]. | |||||||||
履歴 |
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-構造の表示
添付画像 |
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-ダウンロードとリンク
-EMDBアーカイブ
マップデータ | emd_31600.map.gz | 372.4 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-31600-v30.xml emd-31600.xml | 45 KB 45 KB | 表示 表示 | EMDBヘッダ |
画像 | emd_31600.png | 68.6 KB | ||
Filedesc metadata | emd-31600.cif.gz | 16 KB | ||
その他 | emd_31600_additional_1.map.gz | 263.6 MB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-31600 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-31600 | HTTPS FTP |
-検証レポート
文書・要旨 | emd_31600_validation.pdf.gz | 594.1 KB | 表示 | EMDB検証レポート |
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文書・詳細版 | emd_31600_full_validation.pdf.gz | 593.7 KB | 表示 | |
XML形式データ | emd_31600_validation.xml.gz | 7.8 KB | 表示 | |
CIF形式データ | emd_31600_validation.cif.gz | 9.1 KB | 表示 | |
アーカイブディレクトリ | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-31600 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-31600 | 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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「今月の分子」の関連する項目 |
-マップ
ファイル | ダウンロード / ファイル: emd_31600.map.gz / 形式: CCP4 / 大きさ: 512 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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注釈 | composite map for the Xenopus laevie CR subunit. | ||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.387 Å | ||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||
詳細 | EMDB XML:
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-添付データ
-追加マップ: local refined map for the core region of...
ファイル | emd_31600_additional_1.map | ||||||||||||
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注釈 | local refined map for the core region of the Xenopus laevis CR subunit | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-試料の構成要素
+全体 : Xenopus laevis Nuclear Pore Complex (NPC)
+超分子 #1: Xenopus laevis Nuclear Pore Complex (NPC)
+分子 #1: MGC83295 protein
+分子 #2: Nuclear pore complex protein Nup85
+分子 #3: MGC154553 protein
+分子 #4: Nucleoporin SEH1-B
+分子 #5: outer Nup160
+分子 #6: MGC83926 protein
+分子 #7: Nuclear pore complex protein Nup98-Nup96
+分子 #8: Protein SEC13 homolog
+分子 #9: Nuclear pore complex protein
+分子 #10: nuclear pore complex protein Nup133
+分子 #11: Nup358 complex, clamps
+分子 #12: Nuclear pore complex protein Nup93
+分子 #13: Nup155-prov protein
+分子 #14: Nup98
+分子 #15: Nucleoporin Nup88A
+分子 #16: outer Nup133
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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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 |
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凍結 | 凍結剤: ETHANE |
-電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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撮影 | フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 平均電子線量: 75.0 e/Å2 |
電子線 | 加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN |
電子光学系 | 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / 最大 デフォーカス(公称値): 3.0 µm / 最小 デフォーカス(公称値): 1.5 µm |
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
-画像解析
初期モデル | モデルのタイプ: EMDB MAP EMDB ID: |
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最終 再構成 | 想定した対称性 - 点群: C1 (非対称) / 解像度のタイプ: BY AUTHOR / 解像度: 3.7 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 使用した粒子像数: 1279270 |
初期 角度割当 | タイプ: MAXIMUM LIKELIHOOD |
最終 角度割当 | タイプ: MAXIMUM LIKELIHOOD |
-原子モデル構築 1
精密化 | 空間: REAL |
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得られたモデル | PDB-7fik: |