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基本情報
登録情報 | データベース: PDB / ID: 7fik | ||||||
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タイトル | The cryo-EM structure of the CR subunit from X. laevis NPC | ||||||
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![]() | STRUCTURAL PROTEIN / CR / NPC / nucleoporin / Nup358 / Nup93 / Y complex | ||||||
機能・相同性 | ![]() 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 / ribosomal large subunit export from nucleus / mRNA transport / cellular response to nutrient levels / mRNA export from nucleus / nuclear pore / ribosomal small subunit export from nucleus / positive regulation of TORC1 signaling / cellular response to amino acid starvation / GTPase activator activity / nuclear periphery / 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 / cytosol / cytoplasm 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 / クライオ電子顕微鏡法 / 解像度: 3.7 Å | ||||||
![]() | Shi, Y. / Huang, G. / Zhan, X. | ||||||
資金援助 | ![]()
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![]() | ![]() 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]. | ||||||
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構造ビューア | 分子: ![]() ![]() |
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-関連構造データ
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リンク
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集合体
登録構造単位 | ![]()
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要素
-タンパク質 , 12種, 23分子 AaCcDdEeFfHhIiKLMNOWXYj
#1: タンパク質 | 分子量: 227854.141 Da / 分子数: 2 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: Q642R6 #3: タンパク質 | 分子量: 41744.512 Da / 分子数: 2 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: Q05AW3 #4: タンパク質 | 分子量: 39798.602 Da / 分子数: 2 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: Q6GNF1 #5: タンパク質 | 分子量: 162658.234 Da / 分子数: 2 / 由来タイプ: 天然 由来: (天然) ![]() #6: タンパク質 | 分子量: 36588.625 Da / 分子数: 2 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: Q66IZ6 #8: タンパク質 | 分子量: 35361.309 Da / 分子数: 2 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: Q6GNX0 #9: タンパク質 | 分子量: 105398.547 Da / 分子数: 2 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: A2RV69 #11: タンパク質 | 分子量: 322784.344 Da / 分子数: 5 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: A0A1L8HGL2 #13: タンパク質 | | 分子量: 154922.422 Da / 分子数: 1 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: Q7ZWL0 #14: タンパク質 | | 分子量: 13719.902 Da / 分子数: 1 / 由来タイプ: 天然 詳細: The putative protein Nup98 was from the same gene of the chains g/G, whose corresponding peptide is post-translationally cleaved into two separate proteins. 由来: (天然) ![]() #15: タンパク質 | | 分子量: 82570.148 Da / 分子数: 1 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: Q707N0 #16: タンパク質 | | 分子量: 127551.250 Da / 分子数: 1 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: A0A1L8H1I9 |
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-Nuclear pore complex protein ... , 4種, 9分子 BbGgJPSsp
#2: タンパク質 | 分子量: 75160.047 Da / 分子数: 2 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: Q68FJ0 #7: タンパク質 | 分子量: 191067.188 Da / 分子数: 2 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: A0A1L8HBE3 #10: タンパク質 | | 分子量: 111296.109 Da / 分子数: 1 / 由来タイプ: 天然 詳細: This entity was identical with chain j. However, N-terminal region cannot be assigned due to disorder. 由来: (天然) ![]() 参照: UniProt: A0A1L8H1I9 #12: タンパク質 | 分子量: 93565.156 Da / 分子数: 4 / 由来タイプ: 天然 由来: (天然) ![]() 参照: UniProt: Q7ZX96 |
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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: Xenopus laevis Nuclear Pore Complex (NPC) / タイプ: COMPLEX / Entity ID: #1-#13, #16, #15, #14 / 由来: NATURAL |
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由来(天然) | 生物種: ![]() |
緩衝液 | pH: 7.5 |
試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES |
急速凍結 | 凍結剤: ETHANE |
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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: ![]() |
電子レンズ | モード: BRIGHT FIELD / 最大 デフォーカス(公称値): 3000 nm / 最小 デフォーカス(公称値): 1500 nm |
撮影 | 電子線照射量: 75 e/Å2 / フィルム・検出器のモデル: GATAN K3 (6k x 4k) |
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解析
CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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対称性 | 点対称性: C1 (非対称) |
3次元再構成 | 解像度: 3.7 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 1279270 / 対称性のタイプ: POINT |
原子モデル構築 | 空間: REAL |