ジャーナル: J Struct Biol / 年: 2012 タイトル: Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency. 著者: Benjamin E Bammes / Ryan H Rochat / Joanita Jakana / Dong-Hua Chen / Wah Chiu / 要旨: One limitation in electron cryo-microscopy (cryo-EM) is the inability to recover high-resolution signal from the image-recording media at the full-resolution limit of the transmission electron ...One limitation in electron cryo-microscopy (cryo-EM) is the inability to recover high-resolution signal from the image-recording media at the full-resolution limit of the transmission electron microscope. Direct electron detection using CMOS-based sensors for digitally recording images has the potential to alleviate this shortcoming. Here, we report a practical performance evaluation of a Direct Detection Device (DDD®) for biological cryo-EM at two different microscope voltages: 200 and 300 kV. Our DDD images of amorphous and graphitized carbon show strong per-pixel contrast with image resolution near the theoretical sampling limit of the data. Single-particle reconstructions of two frozen-hydrated bacteriophages, P22 and ε15, establish that the DDD is capable of recording usable signal for 3D reconstructions at about 4/5 of the Nyquist frequency, which is a vast improvement over the performance of conventional imaging media. We anticipate the unparalleled performance of this digital recording device will dramatically benefit cryo-EM for routine tomographic and single-particle structural determination of biological specimens.
pH: 7.6 / 詳細: 50 mM Tris pH 7.6, 25 mM NaCl, 2mM EDTA
グリッド
詳細: 400 mesh copper grid
凍結
凍結剤: ETHANE / チャンバー内湿度: 95 % / チャンバー内温度: 170 K / 装置: OTHER / 詳細: Vitrification instrument: Vitrobot / 手法: Blot for 2 seconds before plunging
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電子顕微鏡法
顕微鏡
JEOL 2010F
温度
最低: 100 K / 最高: 100 K / 平均: 100 K
アライメント法
Legacy - 非点収差: objective lens astigmatism was corrected at 100,000 times magnification
日付
2010年6月10日
撮影
カテゴリ: CCD フィルム・検出器のモデル: DIRECT ELECTRON DE-12 (4k x 3k) デジタル化 - サンプリング間隔: 6 µm / 実像数: 200 / 平均電子線量: 17 e/Å2 詳細: Images were collected on a CMOS type detector DE-12 from Direct Electron
試料ホルダー: Side Entry / 試料ホルダーモデル: GATAN LIQUID NITROGEN
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画像解析
CTF補正
詳細: Each Micrograph
最終 再構成
アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 8.5 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ソフトウェア - 名称: MPSA 詳細: Resolution was calculated to be 8.5 Angstroms at 0.5 FSC and 7.3 Angstroms at 0.143 FSC as compared against the 3.8 Angstrom reconstruction of P22. 使用した粒子像数: 7500