National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)
R35 GM122588
米国
引用
ジャーナル: Structure / 年: 2019 タイトル: Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size. 著者: Michael W Martynowycz / Wei Zhao / Johan Hattne / Grant J Jensen / Tamir Gonen / 要旨: Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation ...Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.
凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER 最高温度: 90 K / 最低温度: 77 K
撮影
平均露光時間: 2 sec. / 電子線照射量: 0.04 e/Å2 / フィルム・検出器のモデル: FEI CETA (4k x 4k) / Num. of diffraction images: 100 / 撮影したグリッド数: 1 詳細: Continuous rotation from -30 to +30 with a rotation rate of 0.2 degrees per second and a readout every 3 seconds.