TECHNOLOGY DEVELOPMENT FOR CRYO-EM APPLICATIONS

 描述（由申请人提供）：冷冻电子显微镜（cryo-EM）的最新技术进步使天然状态（未固定、未染色）大分子和细胞的分辨率达到前所未有的水平。氨基酸侧链现在可以在分离的大分子图谱中识别，而不需要结晶。细胞内重要大分子的原位形状及其细胞间的相互作用，现在可以通过冷冻EM断层扫描以低至8.5 μ m的分辨率进行研究。这些进展有望深入了解细胞和分子功能的细节，这是以前从未有过的，推动了生物医学研究的所有领域; NIH通过一项旨在扩大这项技术的使用范围的新RFA（GM-16-001）认识到了它们的重要性。然而，随着人们意识到这项技术的全部前景目前仅适用于某些大分子或细胞类型，目前的热情将减弱。PI的实验室专注于两个项目，这将扩大先进的冷冻EM的适用性：（1）冷冻EM相位板成像和（2）冷冻聚焦离子束（冷冻FIB）准备冷冻EM断层扫描。 在分离的大分子的冷冻-EM中的最高分辨率需要足够大小的分子（> ca. 100 kDa），有限的灵活性，理想情况下具有一定的对称性， 一个相当单一的群体。在不增加电子束损伤的情况下，相位板成像大大提高了对比度，从而可以使用更少的总粒子来完成粒子的充分选择、分选和对准。这将带我们沿着“最后一英里” 更广泛地应用改进的单粒子方法。我们将继续评估各种各样的相移设备，并改进它们在各种“现实世界”情况下的使用。 原位大分子的最高分辨率是通过冷冻EM层析成像，然后通过分子图案的子层析成像平均来实现的。样品必须很薄;平板培养的细胞以前只在它们的边缘进行检查。对于组织中的细胞，冷冻超薄切片术是唯一的选择，这是一种困难且容易产生伪影的技术。我们引入了cryo-FIB研磨，这可以促进所有细胞类型的高分辨率结果。但尽管 尽管最近取得了一些进展，但这仍然是一项极其困难的技术，迄今为止世界上只有很少的实验室实现了这一技术。此外，靶向特定的亚细胞结构是有问题的。如果要实现冷冻FIB铣削用于冷冻EM断层扫描的承诺，必须对其进行改进以扩大其适用性并使其更易于使用。冷冻-EM层析成像也将大大受益于冷冻-EM相位板成像;更好的对比度将提高倾斜系列和子层析成像对准精度，并将揭示弱的精细结构。 我们的实验室已经建立了专门针对我们项目的世界一流的仪器和基础设施（可能是其他地方无法比拟的），我们的工作人员在推进尖端生物电子显微镜方面拥有多年的经验。通过MIRA概念的资金，我们的目标是使我们目前的项目取得圆满成功，同时保持在一个位置，以遵循令人兴奋的新发展。

 DESCRIPTION (provided by applicant): Recent technological advances in cryo-electron microscopy (cryo-EM) have led to unprecedented resolution for native-state (unfixed, unstained) macromolecules and cells. Amino-acid side-chains can now be identified in maps of isolated macromolecules, without the need for crystallization. The in-situ shapes of important macromolecules within cells, and their cellular interaction, can now be studied at down to 8.5 Å resolution by cryo-EM tomography. These advances promise insight into details of cellular and molecular function that have never before been accessible, advancing all fields of biomedical research; NIH has recognized their importance by a new RFA (GM-16-001) that aims to widen access to this technology. However, current enthusiasm will wane with the realization that the full promise of this technology is currently only achieved for certain macromolecules or cell types. The PI's lab focuses on two projects that will widen applicability of advanced cryo-EM: (1) cryo-EM phase-plate imaging and (2) cryo-focused-ion-beam (cryo-FIB) preparation for cryo-EM tomography. The highest resolution in cryo-EM of isolated macromolecules requires molecules of sufficient size (> ca. 100 kDa), limited flexibility, ideally with some symmetry, and which form a fairly homogenous population. Without increasing electron-beam damage, phase-plate imaging greatly improves contrast, so that adequate selection, sorting, and alignment of particles can be accomplished, using fewer total particles. This will take us along the "last mile" to a wider application of the improved single-particle approach. We will continue to evaluate a wide variety of phase-shifting devices, and improve their use in a variety of "real-world" situations. The highest resolution for in-situ macromolecules is achieved by cryo-EM tomography followed by sub-tomogram averaging of molecular motifs. Samples must be thin; flat-cultured cells were previously examined only at their margins. For cells in tissue, cryo-ultramicrotomy, a difficult and artifact-prone technique, was the only choice. We introduced cryo-FIB milling, which can facilitate high-resolution results with all cell types. But in spite of recent progress, this remains an extremely difficult technique, so far implemented in only very few labs in the world. In addition, targeting specific sub-cellular structures is problematical. Ifthe promise of cryo-FIB milling for cryo-EM tomography is to be realized, it must be refined to widen its applicability and make it easier to use. Cryo-EM tomography will also greatly benefit from cryo-EM phase-plate imaging; better contrast will improve tilt-series and sub-tomogram alignment accuracy, and will reveal weak fine structure. Our lab has built up world-class instrumentation and infrastructure specific to our projects (likely unmatched elsewhere), and we have personnel with many years of experience in advancing cutting-edge biological electron microscopy. With funding via the MIRA concept, we aim to bring our current projects to full fruition, while remaining in a position to follow exciting new developments.