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

项目总结 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生物学的一个主要目标，对于了解这些分子在生物医学中扮演的不同角色至关重要。冷冻电子断层成像(CRYO-ET)与亚体积平均(SVA)相结合是目前唯一能够以纳米分辨率在其未受干扰的自然环境中可视化大分子的成像技术。然而，大多数成功的研究都是关于大的络合物或有序的组装，而且分辨率太低，无法揭示分子水平的相互作用。这一技术开发项目的总体目标是设计计算工具，以提高冷冻-ET/SVA的分辨率，并将其适用于更广泛类别的生物医学相关靶标。具体目标是：(1)我们将制定策略，以提高从低剂量断层扫描倾斜系列确定倾斜对比传递函数(CTF)的准确性，(2)我们将设计算法，以提高图像对齐、重建和分类的精度，以减少与数据处理相关的计算B因素，以及(3)我们将把冷冻-ET/SVA的适用范围扩大到更广泛的靶点，包括目前被认为低于该技术技术能力的低分子蛋白质。作为原则的证明，我们将证明我们的方法在可实现的分辨率方面提高了最先进的水平，并可用于在近原子分辨率下确定小的300 kDa酶的结构，这是该领域的一个重要里程碑。我们的研究具有创新性，因为它寻求克服低温技术中的基本技术挑战，以实现这一新兴成像技术的全部潜力。这一建议具有重要意义，因为它表明可以使用冷冻-ET技术以高分辨率可视化具有挑战性的目标，这表明该技术是原位研究重要生物分子的最有前途的途径。最终，通过缩小在体外研究样品的策略和在天然细胞环境中研究蛋白质的技术之间的分辨率差距，我们的方法将允许以前所未有的详细程度可视化处于其功能状态的蛋白质。

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 visualizing macromolecules within their unperturbed native environment at nanometer resolutions. Most successful studies, however, have been of large complexes or ordered 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 (CTF) determination from low-dose tomographic tilt-series, (2) we will design algorithms to improve the accuracy of image alignment, reconstruction and classification aimed at reducing the computational B-factors associated with data processing, and (3) we will extend the applicability of cryo-ET/SVA to a broader class of targets including low molecular weight proteins that are currently considered below the technological capabilities of the technique. As proof of principle, we will demonstrate that our methods improve the state-of-the-art in terms of achievable resolution and can be used to determine the structure of a small 300kDa enzyme at near-atomic resolution, representing a significant milestone 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 demonstrates that challenging targets can be visualized al high-resolution using cryo-ET, indicating that this technique is the most promising route for studying important biomolecules in-situ. Ultimately, by closing the resolution gap between strategies for studying samples in-vitro and techniques to study proteins in their native cellular environment, our methods will allow the visualization of proteins in their functional state at an unprecedented level of detail.