+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-4483 | |||||||||
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タイトル | Correlative FM and ET of GFP-Bax in HeLa cells | |||||||||
マップデータ | Reconstructed tomogram of mitochondria in HeLa cells overexpressing GFP-Bax. | |||||||||
試料 |
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生物種 | Homo sapiens (ヒト) | |||||||||
手法 | 電子線トモグラフィー法 / ネガティブ染色法 | |||||||||
データ登録者 | Ader NR / Hoffmann PC / Ganeva I / Borgeaud AC / Wang C / Youle RJ / Kukulski W | |||||||||
引用 | ジャーナル: Elife / 年: 2019 タイトル: Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis. 著者: Nicholas R Ader / Patrick C Hoffmann / Iva Ganeva / Alicia C Borgeaud / Chunxin Wang / Richard J Youle / Wanda Kukulski / 要旨: During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing ...During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing it. However, it remains unclear how outer membrane openings form. Here, we combined different correlative microscopy and electron cryo-tomography approaches to visualize the effects of Bax activity on mitochondria in human cells. Our data show that Bax clusters localize near outer membrane ruptures of highly variable size. Bax clusters contain structural elements suggesting a higher order organization of their components. Furthermore, unfolding of inner membrane cristae is coupled to changes in the supramolecular assembly of ATP synthases, particularly pronounced at membrane segments exposed to the cytosol by ruptures. Based on our results, we propose a comprehensive model in which molecular reorganizations of the inner membrane and sequestration of outer membrane components into Bax clusters interplay in the formation of outer membrane ruptures. EDITORIAL NOTE: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all ...EDITORIAL NOTE: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter). | |||||||||
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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添付画像 |
-ダウンロードとリンク
-EMDBアーカイブ
マップデータ | emd_4483.map.gz | 646.2 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-4483-v30.xml emd-4483.xml | 11.5 KB 11.5 KB | 表示 表示 | EMDBヘッダ |
画像 | emd_4483.png | 139.9 KB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-4483 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-4483 | 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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-マップ
ファイル | ダウンロード / ファイル: emd_4483.map.gz / 形式: CCP4 / 大きさ: 1.3 GB / タイプ: IMAGE STORED AS SIGNED INTEGER (2 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | Reconstructed tomogram of mitochondria in HeLa cells overexpressing GFP-Bax. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 11.02 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
-試料の構成要素
-全体 : HeLa (homo sapiens)
全体 | 名称: HeLa (homo sapiens) |
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要素 |
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-超分子 #1: HeLa (homo sapiens)
超分子 | 名称: HeLa (homo sapiens) / タイプ: cell / ID: 1 / 親要素: 0 詳細: Cryofixation of cell was performed 16 h after transfection of GFP-Bax plasmid. |
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由来(天然) | 生物種: Homo sapiens (ヒト) |
-実験情報
-構造解析
手法 | ネガティブ染色法 |
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解析 | 電子線トモグラフィー法 |
試料の集合状態 | cell |
-試料調製
緩衝液 | pH: 8.4 / 構成要素 - 名称: PBS |
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染色 | タイプ: POSITIVE / 材質: Uranyl Acetate / 詳細: 0.008% uranyl acetate in acetone |
糖包埋 | 材質: Lowicryl HM20 詳細: We use a temperature-controlling Leica AFS2 with FSP. FS is performed at -90 degrees C for 24-36 h in 0.008 percent (w/v) uranyl acetate in glass-distilled acetone. The temperature is then ...詳細: We use a temperature-controlling Leica AFS2 with FSP. FS is performed at -90 degrees C for 24-36 h in 0.008 percent (w/v) uranyl acetate in glass-distilled acetone. The temperature is then increased to -45 C (5 degrees C/h). Next, the samples are washed three times with acetone and infiltrated with increasing concentrations (10 percent , 25 percent, 50 percent, 75 percent, 2 h each) of Lowicryl HM20 in acetone. During the final mix, the temperature is raised to -35 degrees C (2.5 degree C/h). The temperature is then raised further to -25 degrees C (2.5 degrees C/h), while 100 percent Lowicryl is exchanged three times in 4 h steps with agitation. Then, UV light is applied for 24 h to initialize Lowicryl polymerization. The temperature is then raised to 20 degrees C (5 degrees C/h). At this point, samples can be taken out of the AFS2, but we wait at least 2 days before removing blocks from the plastic wheel to ensure complete polymerization. |
グリッド | 材質: COPPER / メッシュ: 200 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: CONTINUOUS |
加圧凍結法 | 装置: OTHER 詳細: HPF is accomplished using a Leica HPM100 in the provided temperature- and humidity-controlled chamber. To high-pressure freeze cells, we use a carrier method described in the manual for the ...詳細: HPF is accomplished using a Leica HPM100 in the provided temperature- and humidity-controlled chamber. To high-pressure freeze cells, we use a carrier method described in the manual for the Leica EM HPM100 CLEM 3 mm system for HPF of sapphire disks. In brief, carriers are assembled as follows between two plastic half cylinders: (1) 6 mm copper gold-plated support ring in a 6 mm CLEM middle plate, (2) a 3 mm sapphire (cells up), (3) a 3 mm spacer ring, (4) a clean sapphire, and (5) a 6 mm cover ring.. The value given for _emd_high_pressure_freezing.instrument is Leica EM HP100. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER', 'LEICA EM HPM100', 'BAL-TEC HPM 010']) so OTHER is written into the XML file. |
切片作成 | ウルトラミクロトーム - 装置: Ultracut E Microtome (Reichert) ウルトラミクロトーム - 温度: 296 K / ウルトラミクロトーム - 最終 厚さ: 300 nm |
位置合わせマーカー | Manufacturer: Agar Scientific Ltd. / 直径: 15 nm |
-電子顕微鏡法
顕微鏡 | FEI TECNAI F20 |
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電子線 | 加速電圧: 200 kV / 電子線源: FIELD EMISSION GUN |
電子光学系 | C2レンズ絞り径: 50.0 µm / 照射モード: SPOT SCAN / 撮影モード: BRIGHT FIELDBright-field microscopy |
試料ステージ | 試料ホルダーモデル: OTHER |
詳細 | ET was done in STEM mode on an axial brightfield detector with a high-tilt tomography holder (Model 2020; Fischione Instruments) at a 1.1nm pixel size with a camera length of 200 mm. |
撮影 | フィルム・検出器のモデル: OTHER / デジタル化 - サイズ - 横: 2048 pixel / デジタル化 - サイズ - 縦: 2048 pixel / 平均電子線量: 1000.0 e/Å2 詳細: ET was done in STEM mode on an axial brightfield detector with a high-tilt tomography holder (Model 2020; Fischione Instruments) at a 1.1nm pixel size with a camera length of 200 mm. [NOTE: ...詳細: ET was done in STEM mode on an axial brightfield detector with a high-tilt tomography holder (Model 2020; Fischione Instruments) at a 1.1nm pixel size with a camera length of 200 mm. [NOTE: Electron dose unknown. Value specified here is a dummy] |
実験機器 | モデル: Tecnai F20 / 画像提供: FEI Company |
-画像解析
最終 再構成 | アルゴリズム: BACK PROJECTION / ソフトウェア - 名称: eTomo (ver. 4.10.20) / 詳細: 237 tilted images used from two axies / 使用した粒子像数: 237 |
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