A near-atomic resolution structure of the mouse voltage dependent anion channel (mVDAC) is determined by combining cryogenic focused ion-beam (FIB) milling and microcrystal electron diffraction (MicroED). The crystals were grown in a viscous modified bicelle suspension which limited their size and made them unsuitable for conventional X-ray crystallography. Individual thin, plate-like crystals were identified using scanning electron microscopy (SEM) and focused ion-beam (FIB) imaging at high magnification. Three crystals were milled into thin lamellae. MicroED data were collected from each lamellae and merged to increase completeness. Unmodelled densities were observed between protein monomers, suggesting the presence of lipids that likely mediate crystal contacts. This work demonstrates the utility of milling membrane protein microcrystals grown in viscous media using a focused ion-beam for subsequent structure determination by MicroED for samples that are not otherwise tractable by other crystallographic methods. To our knowledge, the structure presented here is the first of a membrane protein crystallized in a lipid matrix and solved by MicroED.

中文翻译：

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脂质包裹的哺乳动物线粒体电压依赖性阴离子通道的MicroED结构

通过组合低温聚焦离子束（FIB）铣削和微晶电子衍射（MicroED）来确定小鼠电压依赖性阴离子通道（mVDAC）的近原子分辨率结构。晶体在粘性改性的比塞勒悬浮液中生长，这限制了它们的尺寸，使其不适合常规X射线晶体学。使用扫描电子显微镜（SEM）和聚焦离子束（FIB）成像以高放大倍数识别出单个薄板状晶体。将三个晶体研磨成薄片。从每个薄片收集MicroED数据并合并以提高完整性。在蛋白质单体之间观察到未建模的密度，表明存在可能介导晶体接触的脂质。这项工作证明了使用聚焦离子束研磨在粘性介质中生长的膜蛋白微晶体用于随后通过MicroED确定其他晶体方法无法处理的样品的结构的实用性。据我们所知，这里介绍的结构是在脂质基质中结晶并通过MicroED解析的膜蛋白的第一个。