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- EMDB-7321: Integrative Structure and Functional Anatomy of a Nuclear Pore Complex -
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データを開く
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基本情報
登録情報 | データベース: EMDB / ID: EMD-7321 | |||||||||||||||||||||||||||||||||||||||
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タイトル | Integrative Structure and Functional Anatomy of a Nuclear Pore Complex | |||||||||||||||||||||||||||||||||||||||
![]() | Integrative Structure and Functional Anatomy of a Nuclear Pore Complex | |||||||||||||||||||||||||||||||||||||||
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生物種 | ![]() ![]() | |||||||||||||||||||||||||||||||||||||||
手法 | サブトモグラム平均法 / クライオ電子顕微鏡法 / 解像度: 28.0 Å | |||||||||||||||||||||||||||||||||||||||
![]() | Kim SJ / Fernandez-Martinez J / Nudelman I / Shi Y / Zhang W / Ludtke SJ / Akey CW / Chait BT / Sali A / Rout MP | |||||||||||||||||||||||||||||||||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Integrative structure and functional anatomy of a nuclear pore complex. 著者: Seung Joong Kim / Javier Fernandez-Martinez / Ilona Nudelman / Yi Shi / Wenzhu Zhang / Barak Raveh / Thurston Herricks / Brian D Slaughter / Joanna A Hogan / Paula Upla / Ilan E Chemmama / ...著者: Seung Joong Kim / Javier Fernandez-Martinez / Ilona Nudelman / Yi Shi / Wenzhu Zhang / Barak Raveh / Thurston Herricks / Brian D Slaughter / Joanna A Hogan / Paula Upla / Ilan E Chemmama / Riccardo Pellarin / Ignacia Echeverria / Manjunatha Shivaraju / Azraa S Chaudhury / Junjie Wang / Rosemary Williams / Jay R Unruh / Charles H Greenberg / Erica Y Jacobs / Zhiheng Yu / M Jason de la Cruz / Roxana Mironska / David L Stokes / John D Aitchison / Martin F Jarrold / Jennifer L Gerton / Steven J Ludtke / Christopher W Akey / Brian T Chait / Andrej Sali / Michael P Rout / ![]() 要旨: Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full ...Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex. | |||||||||||||||||||||||||||||||||||||||
履歴 |
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構造の表示
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構造ビューア | EMマップ: ![]() ![]() ![]() |
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 | ![]() | 18.9 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 20.6 KB 20.6 KB | 表示 表示 | ![]() |
画像 | ![]() | 271.1 KB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
類似構造データ | |
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電子顕微鏡画像生データ | ![]() Data #1: Unaligned K2 DED image tilt series of yeast NPC and associated files [micrographs - single frame]) ![]() Data size: 11.8 Data #1: Raw micrographs of Nic96 complex [micrographs - single frame] Data #2: Sc Nic96 complex class averages [class averages]) |
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リンク
EMDBのページ | ![]() ![]() |
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マップ
ファイル | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | Integrative Structure and Functional Anatomy of a Nuclear Pore Complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 5.3 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
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-添付データ
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試料の構成要素
-全体 : Saccharomyces cerevisiae NPC (nuclear pore complex)
全体 | 名称: Saccharomyces cerevisiae NPC (nuclear pore complex) |
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要素 |
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-超分子 #1: Saccharomyces cerevisiae NPC (nuclear pore complex)
超分子 | 名称: Saccharomyces cerevisiae NPC (nuclear pore complex) / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1 / 詳細: Affinity-purified isolated whole NPCs |
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由来(天然) | 生物種: ![]() ![]() |
分子量 | 実験値: 87 MDa |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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![]() | サブトモグラム平均法 |
試料の集合状態 | particle |
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試料調製
濃度 | 0.3 mg/mL | ||||||||||||||||||||||||
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緩衝液 | pH: 7.4 構成要素:
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グリッド | モデル: Quantifoil R2/1 / 材質: COPPER / メッシュ: 300 詳細: A medium thick carbon film was used to support the NPCs over the holes. Before use, the grids were glow discharged in air, floated on 5 uL sample drops for 45 minutes and then washed by ...詳細: A medium thick carbon film was used to support the NPCs over the holes. Before use, the grids were glow discharged in air, floated on 5 uL sample drops for 45 minutes and then washed by serial transfer on 4 x 20 micro-litre drops of sample buffer without glycerol. | ||||||||||||||||||||||||
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 293 K / 装置: FEI VITROBOT MARK III 詳細: Buffer on the grid was removed by blotting from the bottom with a tool that held a filter paper wedge, using access through the left-hand port. Then 2 micro-litre of freezing buffer was added ...詳細: Buffer on the grid was removed by blotting from the bottom with a tool that held a filter paper wedge, using access through the left-hand port. Then 2 micro-litre of freezing buffer was added to the grid from the right-hand port and the grid was plunge frozen in liquid ethane after blotting.. | ||||||||||||||||||||||||
詳細 | Sample isolated in one affinity step - pullout from frozen yeast cell grindate. |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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特殊光学系 | 球面収差補正装置: Titan-Krios equipped with a spherical aberration (Cs) corrector 色収差補正装置: none / 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 - エネルギー下限: 1 eV エネルギーフィルター - エネルギー上限: 20 eV |
詳細 | The electron gun was an XFEG. A total of 253 tilt series were collected in steps between -60, 0 and 60 degrees in increments of 2.5 - 4 degrees for different tilt series. While the full tilt range was used for tomogram reconstruction, in the final subtomogram averaging step only data up to +/-45 degrees tilt from each sub-volume were included in the final average. A defocus range of -4.6 to -7.5 microns was used. |
撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: INTEGRATING / デジタル化 - サイズ - 横: 3838 pixel / デジタル化 - サイズ - 縦: 3710 pixel / 撮影したグリッド数: 1 / 平均電子線量: 3.0 e/Å2 詳細: The dose target for each tilt series was 90-100 electrons/angstrom squared and followed a cosine alpha dose curve with a flux of 20 electrons/pixel/second, and a dose of 3.5 ...詳細: The dose target for each tilt series was 90-100 electrons/angstrom squared and followed a cosine alpha dose curve with a flux of 20 electrons/pixel/second, and a dose of 3.5 electrons/angstrom squared for the zero tilt image. |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | 倍率(補正後): 9434 / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 0.001 mm / 最大 デフォーカス(公称値): 7.5 µm / 最小 デフォーカス(公称値): 4.6 µm |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER ホルダー冷却材: NITROGEN |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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画像解析
最終 再構成 | 想定した対称性 - 点群: C8 (8回回転対称) / アルゴリズム: FOURIER SPACE / 解像度のタイプ: BY AUTHOR / 解像度: 28.0 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: EMAN2 (ver. 2.1) 詳細: The final map has been fully CTF corrected and filtered based on the estimated local resolution. 使用したサブトモグラム数: 1864 |
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抽出 | トモグラム数: 121 / 使用した粒子像数: 6416 / 手法: manual selection / ソフトウェア - 名称: EMAN2 (ver. 2.12) / ソフトウェア - 詳細: e2spt_boxer.py 詳細: A low pass filter of 100 Angstroms was applied to binned 3X SIRT tomograms prior to particle picking. Final unbinned sub-volumes of 300 x 300 x 300 were extracted from the original tomograms. |
CTF補正 | ソフトウェア - 名称: EMAN2 (ver. 2.1) 詳細: Phase flipping of tilted images with etomo in IMOD running in batchtomo mode. The final reconstruction used 1,864 (of the 6,416 initial) particles. Theoretical CTF curves for the mean defocus ...詳細: Phase flipping of tilted images with etomo in IMOD running in batchtomo mode. The final reconstruction used 1,864 (of the 6,416 initial) particles. Theoretical CTF curves for the mean defocus values present in the tomograms were averaged assuming 10% amplitude contrast. The reciprocal of this curve was then applied as a filter to the final uncorrected map. |
最終 角度割当 | タイプ: OTHER / ソフトウェア - 名称: EMAN2 ソフトウェア - 詳細: e2spt_classaverage.py in theEMAN2 single particle tomography package 詳細: Iterative 3D alignment of sub-volumes to a reference volume, using the C8 symmetry of the NPC. |
-原子モデル構築 1
詳細 | The integrative structure modeling protocol was scripted using the Python Modeling Interface (PMI) package, version 4d97507, a library for modeling macromolecular complexes based on our open-source Integrative Modeling Platform (IMP) package, version 2.6 (https://integrativemodeling.org) |
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精密化 | プロトコル: OTHER |