+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 6i5a | ||||||
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タイトル | Tobacco Mosaic Virus | ||||||
要素 | Capsid protein | ||||||
キーワード | VIRUS / TMV / helical virus / coat protein / plant pathogen | ||||||
機能・相同性 | 機能・相同性情報 | ||||||
生物種 | Tobacco mosaic virus (ウイルス) | ||||||
手法 | 電子顕微鏡法 / らせん対称体再構成法 / クライオ電子顕微鏡法 / 解像度: 2.3 Å | ||||||
データ登録者 | Song, B. / Flegler, V. / Makbul, C. / Rasmussen, T. / Bottcher, B. | ||||||
資金援助 | ドイツ, 1件
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引用 | ジャーナル: Ultramicroscopy / 年: 2019 タイトル: Capabilities of the Falcon III detector for single-particle structure determination. 著者: Boyuan Song / Julian Lenhart / Vanessa Judith Flegler / Cihan Makbul / Tim Rasmussen / Bettina Böttcher / 要旨: Direct electron detectors are an essential asset for the resolution revolution in electron cryo microscopy of biological objects. The direct detectors provide two modes of data acquisition; the ...Direct electron detectors are an essential asset for the resolution revolution in electron cryo microscopy of biological objects. The direct detectors provide two modes of data acquisition; the counting mode in which single electrons are counted, and the integrating mode in which the signal that arises from the incident electrons is integrated. While counting mode leads to far higher detective quantum efficiency at all spatial frequencies, the integrating mode enables faster data acquisition at higher exposure rates. For optimal throughput at best possible resolution it is important to understand when the better performance in counting mode becomes essential for solving a structure and when the lower detective quantum efficiency in integrating mode can be compensated by increasing the number of particles in the data set. Here, we provide a case study of the Falcon III camera, which has counting mode capability at exposure rates of <0.9 e/Px² and integrating mode capability at exposure rates above 10 e/Px². We found that counting mode gives better resolution for medium sized complexes such as the β-galactosidase (465 kDa) (2.2 Å, 97% of Nyquist vs. 2.4 Å, 89% of Nyquist) with data sets of similar size. However, for larger particles such as Hepatitis B virus capsid like particles (4.8 MDa) we did not find any resolution gain in counting mode. | ||||||
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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構造ビューア | 分子: MolmilJmol/JSmol |
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 6i5a.cif.gz | 41.4 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb6i5a.ent.gz | 27.9 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 6i5a.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/i5/6i5a ftp://data.pdbj.org/pub/pdb/validation_reports/i5/6i5a | HTTPS FTP |
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-関連構造データ
-リンク
-集合体
登録構造単位 |
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1 |
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-要素
#1: タンパク質 | 分子量: 17636.621 Da / 分子数: 1 / 由来タイプ: 天然 由来: (天然) Tobacco mosaic virus (strain vulgare) (ウイルス) 株: vulgare / 参照: UniProt: P69687 |
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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: HELICAL ARRAY / 3次元再構成法: らせん対称体再構成法 |
-試料調製
構成要素 | 名称: Tobacco mosaic virus / タイプ: VIRUS / Entity ID: all / 由来: NATURAL |
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分子量 | 値: 40 MDa / 実験値: NO |
由来(天然) | 生物種: Tobacco mosaic virus (ウイルス) |
ウイルスについての詳細 | 中空か: NO / エンベロープを持つか: NO / 単離: STRAIN / タイプ: VIRION |
天然宿主 | 生物種: Nicotiana sp. |
ウイルス殻 | 直径: 180 nm |
緩衝液 | pH: 7 |
試料 | 濃度: 10 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES |
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 400 divisions/in. / グリッドのタイプ: Quantifoil R1.2/1.3 |
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 277 K 詳細: Quantifoil 400 mesh copper grids R 1.2/1.3 were used (Quantifoil Micro Tools GmbH, Jena, Germany). Grids were glow discharged in air at a pressure of 2.2x10-2 Torr for 2 min at medium power ...詳細: Quantifoil 400 mesh copper grids R 1.2/1.3 were used (Quantifoil Micro Tools GmbH, Jena, Germany). Grids were glow discharged in air at a pressure of 2.2x10-2 Torr for 2 min at medium power with a Harrick Plasma cleaner (PDC-002). Subsequently, 3-4 ul of the sample was pipetted onto the glow discharged grids and plunge frozen in liquid Ethane with a Vitrobot IV (FEI). The sample chamber of the Vitrobot was maintained at 4C and 100% humidity. Wait time between sample application and blotting was 45 s, the drain time 0 s, the blot time 3 s and the blot force 0. |
-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: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 75000 X / 最大 デフォーカス(公称値): 1800 nm / 最小 デフォーカス(公称値): 1000 nm / Calibrated defocus min: 580 nm / 最大 デフォーカス(補正後): 2120 nm / Cs: 2.7 mm / C2レンズ絞り径: 70 µm / アライメント法: COMA FREE |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER 最高温度: 80 K / 最低温度: 79 K |
撮影 | 平均露光時間: 5 sec. / 電子線照射量: 55 e/Å2 / 検出モード: INTEGRATING フィルム・検出器のモデル: FEI FALCON III (4k x 4k) 撮影したグリッド数: 1 / 実像数: 1001 |
画像スキャン | 横: 4096 / 縦: 4096 |
-解析
EMソフトウェア |
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画像処理 | 詳細: Movies were motion corrected and exposure weighted either with motioncor2 (see tables 1-3 for details). The defocus was determined for the motion corrected and exposure weighted averages with ctffind4 | ||||||||||||||||||||||||||||||||||||||||||||||||||
CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
らせん対称 | 回転角度/サブユニット: 22.037 ° / 軸方向距離/サブユニット: 1.39615 Å / らせん対称軸の対称性: C1 | ||||||||||||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 393692 詳細: For TMV, some helices were picked manually with the helix picker implemented in relion 2.1. An initial 3D model of these segments was determined using the relion helix option with a cylinder ...詳細: For TMV, some helices were picked manually with the helix picker implemented in relion 2.1. An initial 3D model of these segments was determined using the relion helix option with a cylinder as the starting reference. The aligned helical segments after 3D refinement were subjected to 2D classification without alignment. Three of the 2D class averages were selected as templates for automatic picking of the TMV segments in relion. | ||||||||||||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 2.3 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 91927 / アルゴリズム: FOURIER SPACE / クラス平均像の数: 1 / 対称性のタイプ: HELICAL | ||||||||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | B value: 105 / プロトコル: AB INITIO MODEL / 空間: RECIPROCAL |