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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-0732 | |||||||||
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タイトル | Cryo electron tomogram of cryo-lamella of rat skeleton muscle | |||||||||
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生物種 | ![]() ![]() | |||||||||
手法 | 電子線トモグラフィー法 | |||||||||
![]() | Zhang J / Zhang D / Sun L / Sun F | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: VHUT-cryo-FIB, a method to fabricate frozen hydrated lamellae from tissue specimens for in situ cryo-electron tomography. 著者: Jianguo Zhang / Danyang Zhang / Lei Sun / Gang Ji / Xiaojun Huang / Tongxin Niu / Jiashu Xu / Chengying Ma / Yun Zhu / Ning Gao / Wei Xu / Fei Sun / ![]() 要旨: Cryo-electron tomography (cryo-ET) provides a promising approach to study intact structures of macromolecules in situ, but the efficient preparation of high-quality cryosections represents a ...Cryo-electron tomography (cryo-ET) provides a promising approach to study intact structures of macromolecules in situ, but the efficient preparation of high-quality cryosections represents a bottleneck. Although cryo-focused ion beam (cryo-FIB) milling has emerged for large and flat cryo-lamella preparation, its application to tissue specimens remains challenging. Here, we report an integrated workflow, VHUT-cryo-FIB, for efficiently preparing frozen hydrated tissue lamella that can be readily used in subsequent cryo-ET studies. The workflow includes vibratome slicing, high-pressure freezing, ultramicrotome cryo-trimming and cryo-FIB milling. Two strategies were developed for loading cryo-lamella via a side-entry cryo-holder or an FEI AutoGrid. The workflow was validated by using various tissue specimens, including rat skeletal muscle, rat liver and spinach leaf specimens, and in situ structures of ribosomes were obtained at nanometer resolution from the spinach and liver samples. | |||||||||
履歴 |
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構造の表示
ムービー |
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添付画像 |
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マップデータ | ![]() | 648.8 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 13.1 KB 13.1 KB | 表示 表示 | ![]() |
画像 | ![]() | 125.2 KB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
関連構造データ | ![]() 0733C ![]() 0734C C: 同じ文献を引用 ( |
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電子顕微鏡画像生データ | ![]() Data size: 1.3 Data #1: Unaligned tilt series of cryo lamella of rat skeleton muscle [tilt series]) |
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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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ボクセルのサイズ | X=Y=Z: 26.1 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 : cryo-lamella of rat skeleton muscle
全体 | 名称: cryo-lamella of rat skeleton muscle |
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要素 |
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-超分子 #1: cryo-lamella of rat skeleton muscle
超分子 | 名称: cryo-lamella of rat skeleton muscle / タイプ: tissue / ID: 1 / 親要素: 0 |
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由来(天然) | 生物種: ![]() ![]() |
-実験情報
-構造解析
![]() | 電子線トモグラフィー法 |
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試料の集合状態 | tissue |
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試料調製
緩衝液 | pH: 7 / 詳細: phosphate buffered saline (PBS) |
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詳細 | Mice were sacrificed and muscle was removed and cut into small bulks with a razor blade. Then a bulk of muscle was fixed on the specimen holder of VT1200S vibrating blade microtome for slicing. The thickness of each slice was set to 80-100 um. The generated muscle slice was cut into appropriate size for high pressure freezing. |
加圧凍結法 | 装置: OTHER 詳細: Muscle tissue slice was put in the recess of the carrier and cryoprotectant 1-hexadecene was added to fill the surrounding area. Then a sapphire disk was loaded on top of the carrier before ...詳細: Muscle tissue slice was put in the recess of the carrier and cryoprotectant 1-hexadecene was added to fill the surrounding area. Then a sapphire disk was loaded on top of the carrier before the whole composed sandwich was frozen.. The value given for _emd_high_pressure_freezing.instrument is HPF COMPACT 01. This is not in a list of allowed values {'BAL-TEC HPM 010', 'LEICA EM HPM100', 'OTHER', 'LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER'} so OTHER is written into the XML file. |
Cryo protectant | 1-hexadecene |
切片作成 | 集束イオンビーム - 装置: OTHER / 集束イオンビーム - イオン: OTHER / 集束イオンビーム - 電圧: 30 kV / 集束イオンビーム - 電流: 0.08 nA / 集束イオンビーム - 時間: 3600 sec. / 集束イオンビーム - 温度: 93 K / 集束イオンビーム - Initial thickness: 200 nm / 集束イオンビーム - 最終 厚さ: 20 nm 集束イオンビーム - 詳細: Then the carrier was transfer with the cryo-transfer shuttle17 into the SEM chamber by using Quorum PP3000T cryotransfer system under -180 degree. To improve sample ...集束イオンビーム - 詳細: Then the carrier was transfer with the cryo-transfer shuttle17 into the SEM chamber by using Quorum PP3000T cryotransfer system under -180 degree. To improve sample conductivity and reduce curtaining artifacts, the samples were deposited with organometallic platinum using the in situ gas injection system (GIS) operated at 5 seconds gas injection time before milling. During the cryo-FIB milling process, the milling angle is nearly in parallel with the carrier, and the milling was performed parallel from both sides of the sample platform to produce lamella. Rough milling is produced with the accelerating voltage of the ion beam at 30 kV, and current at 0.79 nA-0.43 nA. The initial milling width is about 20 um and depth is about 20 um. To facilitate tomography data collection, ice at the notch above lamella was removed to get a trapezoid-shaped milling pattern. After rough milling, one side of the lamella is jagged from the main platform. When the thickness of lamella reaches about 1 um the ion current is reduced to 0.23 nA or 80 pA until thickness finally reaching 150 to 250 nm.. The value given for _emd_sectioning_focused_ion_beam.instrument is Helios NanoLab 600i. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file. |
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電子顕微鏡法
顕微鏡 | TFS TALOS F200C |
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撮影 | フィルム・検出器のモデル: FEI CETA (4k x 4k) / デジタル化 - サイズ - 横: 4096 pixel / デジタル化 - サイズ - 縦: 4096 pixel / デジタル化 - サンプリング間隔: 14.0 µm / 撮影したグリッド数: 1 / 実像数: 82 / 平均露光時間: 1.0 sec. / 平均電子線量: 3.0 e/Å2 |
電子線 | 加速電圧: 200 kV / 電子線源: ![]() |
電子光学系 | 最大 デフォーカス(補正後): 10.0 µm / 最小 デフォーカス(補正後): 8.0 µm / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 倍率(公称値): 13500 |
試料ステージ | 試料ホルダーモデル: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER ホルダー冷却材: NITROGEN |
実験機器 | ![]() モデル: Tecnai F20 / 画像提供: FEI Company |
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画像解析
最終 再構成 | アルゴリズム: FOURIER SPACE / ソフトウェア - 名称: ICON (ver. 1.2.9) 詳細: ICON version 1.2.9 was used for the final reconstruction and the reconstruction algorithm is iterative compressed-sensing optimized NUFFT. 使用した粒子像数: 43 |
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