A Computational Platform for In-Situ Structure Determination at Near-Atomic Resolution using Cryo-Electron Tomography

项目概要 了解蛋白质如何在细胞内相互作用以执行特定功能是现代科学的一个主要目标 生物学，对于理解这些分子在生物医学中发挥的不同作用至关重要。冷冻电子 断层扫描 (cryo-ET) 与子体积平均 (SVA) 相结合是目前唯一的成像技术 允许在未受干扰的天然环境中以纳米分辨率对大分子进行成像。大多数 然而，成功的研究是针对大型复合物或超分子组装体，并且分辨率较高 太低而无法揭示分子水平的相互作用。本次技术开发的总体目标 该项目的目的是设计计算工具来提高冷冻电子断层扫描/SVA 的分辨率并将其适用性扩展到 更广泛的生物医学相关目标。具体目标是：（1）我们将制定改进策略 从低剂量断层扫描投影确定倾斜对比度传递函数的准确性，(2) 我们 将设计算法以提高子体积对齐、重建和分类的准确性 减少与数据处理相关的计算 B 因素，(3) 我们将优化成像和 数据处理参数，能够对更广泛的目标进行高分辨率研究，包括小型目标 复合物。作为原理证明，我们实现了平台的第一代原型并在其上进行了测试 通过冷冻电子断层扫描（cryo-ET）成像的单分散样品。初步结果表明我们的平台：（1）改进 就可实现的分辨率而言是最先进的，并且 (2) 可用于确定 300kDa 的结构 酶的分辨率为 3.9 Å，代表了该领域的突破性成就。我们的研究具有创新性 因为它寻求克服冷冻电子断层扫描的基本技术挑战，以充分发挥其潜力 这种新兴的成像技术。该提案意义重大，因为它将首次证明低 使用冷冻电子断层扫描 (cryo-ET) 可以以近原子分辨率对分子量目标进行成像，这表明该技术 是重要生物分子原位成像最有前途的途径。最终，通过缩小“分辨率差距” 在高分辨率（X 射线、NMR 和单颗粒冷冻）研究单分散样品的策略之间 EM）和在天然环境中研究蛋白质的技术，我们的方法将允许可视化 以前所未有的详细程度了解其功能状态下的蛋白质复合物。

PROJECT SUMMARY Understanding how proteins interact within the cell to perform specific functions is a major goal of modern biology, and vital for understanding the diverse roles these molecules play in biomedicine. Cryo-electron tomography (cryo-ET) combined with sub-volume averaging (SVA) is currently the only imaging technology that allows imaging macromolecules within their unperturbed native environment at nanometer resolutions. Most successful studies, however, have been of large complexes or supramolecular assemblies, and at resolutions that are too low to reveal molecular level interactions. The overall objective of this Technology Development project is to design computational tools to improve the resolution of cryo-ET/SVA and extend its applicability to a wider class of biomedically relevant targets. The specific aims are: (1) we will develop strategies to improve the accuracy of the tilted contrast transfer function determination from low-dose tomographic projections, (2) we will design algorithms to improve the accuracy of sub-volume alignment, reconstruction and classification aimed at reducing the computational B-factors associated with data processing, and (3) we will optimize imaging and data processing parameters to enable high-resolution studies of a wider class of targets including small complexes. As proof of principle, we implemented a first-generation prototype of our platform and tested it on monodisperse samples imaged by cryo-ET. The preliminary results demonstrate that our platform: (1) improves the state-of-the-art in terms of achievable resolution, and (2) can be used to determine the structure of a 300kDa enzyme at 3.9 Å resolution, representing a ground-breaking achievement for the field. Our research is innovative because it seeks to overcome fundamental technical challenges in cryo-ET needed to realize the full potential of this emerging imaging technology. The proposal is significant because it will be the first demonstration that low- molecular weight targets can be imaged at near-atomic resolution using cryo-ET, indicating that this technique is the most promising route for imaging important biomolecules in-situ. Ultimately, by closing the “resolution gap” between strategies for studying monodisperse samples at high-resolution (X-ray, NMR and single-particle cryo- EM) and techniques to study proteins in their native environments, our methods will allow the visualization of protein complexes in their functional state at unprecedented levels of detail.