Biotechnology and Biological Sciences Research Council (BBSRC)
BB/P020208/1
英国
Biotechnology and Biological Sciences Research Council (BBSRC)
BB/P026397/1
英国
Wellcome Trust
108466/Z/15/Z
英国
引用
ジャーナル: Structure / 年: 2020 タイトル: Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales. 著者: David P Klebl / Molly S C Gravett / Dimitrios Kontziampasis / David J Wright / Robin S Bon / Diana C F Monteiro / Martin Trebbin / Frank Sobott / Howard D White / Michele C Darrow / Rebecca F ...著者: David P Klebl / Molly S C Gravett / Dimitrios Kontziampasis / David J Wright / Robin S Bon / Diana C F Monteiro / Martin Trebbin / Frank Sobott / Howard D White / Michele C Darrow / Rebecca F Thompson / Stephen P Muench / 要旨: A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the ...A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic "silver bullet" for improving the quality of every cryoEM sample preparation.
フィルム・検出器のモデル: FEI FALCON III (4k x 4k) 検出モード: INTEGRATING / 平均露光時間: 1.5 sec. / 平均電子線量: 78.0 e/Å2
実験機器
モデル: Titan Krios / 画像提供: FEI Company
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画像解析
初期 角度割当
タイプ: NOT APPLICABLE
最終 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION 詳細: The final angular assignment was done in a bigger, combined dataset which was then split into the original datasets to generate this reconstruction