Mass-selective and ice-free electron cryomicroscopy protein sample preparation via native electrospray ion-beam deposition.
Mass-selective and ice-free electron cryomicroscopy protein sample preparation via native electrospray ion-beam deposition.
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DOI：
10.1093/pnasnexus/pgac153
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发表时间：
2022-09
期刊：
影响因子：
--
通讯作者：
Rauschenbach, Stephan
中科院分区：
文献类型：
--
作者：
Esser, Tim K.;Bohning, Jan;Fremdling, Paul;Agasid, Mark T.;Costin, Adam;Fort, Kyle;Konijnenberg, Albert;Gilbert, Joshua D.;Bahm, Alan;Makarov, Alexander;Robinson, Carol V.;Benesch, Justin L. P.;Baker, Lindsay;Bharat, Tanmay A. M.;Gault, Joseph;Rauschenbach, Stephan
关键词：
尽管电子冷冻显微镜（cryo-EM）和单颗粒分析（SPA）的样品制备和分类算法取得了巨大进步，但样品的异质性仍然是一个主要挑战，并可能阻碍获得高分辨率结构。此外，对给定样品的制备条件进行优化可能很耗时。在目前的工作中，证明了原生电喷雾离子束沉积（原生ES-IBD）是一种可靠的替代方法，用于制备极高纯度的样品，基于真空中的大量选择。折叠的蛋白质离子由天然电喷雾电离产生，与其他蛋白质、污染物、聚集物和碎片分离，轻轻沉积在冷冻电镜网格上，在液氮中冷冻，随后通过冷冻电镜成像。我们展示了大量选择蛋白质复合物的无冰低温电镜网格的均匀覆盖。SPA显示复合物仍然折叠和组装，但二级和三级结构的变化目前限制了2D类和3D EM密度图的信息。我们确定并讨论需要解决的挑战，以获得与已建立的冷冻电镜工作流程相当的分辨率。我们的研究结果表明，天然ES-IBD有潜力增加冷冻电镜测定蛋白质结构的范围和通量，并提供气相和溶液相蛋白质结构之间的重要联系。
Despite tremendous advances in sample preparation and classification algorithms for electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA), sample heterogeneity remains a major challenge and can prevent access to high-resolution structures. In addition, optimization of preparation conditions for a given sample can be time-consuming. In the current work, it is demonstrated that native electrospray ion-beam deposition (native ES-IBD) is an alternative, reliable approach for the preparation of extremely high-purity samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, separated from other proteins, contaminants, aggregates, and fragments, gently deposited on cryo-EM grids, frozen in liquid nitrogen, and subsequently imaged by cryo-EM. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected protein complexes. SPA reveals that the complexes remain folded and assembled, but variations in secondary and tertiary structures are currently limiting information in 2D classes and 3D EM density maps. We identify and discuss challenges that need to be addressed to obtain a resolution comparable to that of the established cryo-EM workflow. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM for protein structure determination and provide an essential link between gas-phase and solution-phase protein structures.
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影响因子：
15
作者：
Anggara K;Zhu Y;Delbianco M;Rauschenbach S;Abb S;Seeberger PH;Kern K
通讯作者：
Kern K
DOI：
10.1073/pnas.0610313104
发表时间：
2007-06-05
影响因子：
11.1
作者：
Cavalli, Andrea;Salvatella, Xavier;Vendruscolo, Michele
通讯作者：
Vendruscolo, Michele
影响因子：
14.8
作者：
Hernandez, Helena;Robinson, Carol V.
通讯作者：
Robinson, Carol V.
影响因子：
16.6
作者：
Abb S;Harnau L;Gutzler R;Rauschenbach S;Kern K
通讯作者：
Kern K
影响因子：
3.3
作者：
Bakhtiari, Maryam;Konermann, Lars
通讯作者：
Konermann, Lars