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

使用冷冻电子断层扫描以近原子分辨率原位结构测定的计算平台

• 批准号: 10581369
• 负责人: Alberto Bartesaghi
• 金额: $ 21万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10581369
• 关键词: Achievement; Adoption; Algorithm Design; Algorithms; Benchmarking; Biological; Biology; Biomedical Research; Cells; Classification; Communities; Complex; Computer software; Computing Methodologies; Cryo-electron tomography; Cryoelectron Microscopy; Data; Data Collection; Data Set; Development; Discipline; Disease; Dose; Electron Microscope; Emerging Technologies; Environment; Enzymes; Event; Excision; Freezing; Generations; Geometry; Goals; HIV-1; Hybrids; Hydration status; Image; Imaging Techniques; Imaging technology; In Situ; In Vitro; Macromolecular Complexes; Maps; Medical; Methods; Microscopy; Modeling; Modernization; Molecular; Molecular Structure; Molecular Weight; Outcome; Performance; Play; Preparation; Proteins; Research; Resolution; Role; Route; Sampling; Sampling Studies; Series; Specimen; Structure; Techniques; Technology; Testing; Virion; Visualization; Work; X-Ray Crystallography; algorithm development; combat; computational platform; computerized data processing; computerized tools; crystallinity; design; experience; gag Gene Products; high resolution imaging; image processing; improved; innovation; interest; macromolecule; molecular assembly/self assembly; nanometer resolution; novel; overexpression; particle; protein complex; protein structure; public health relevance; reconstitution; reconstruction; structural biology; technology development; three dimensional structure; tomography; tool

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.

项目摘要 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生物学的主要目标，对于理解这些分子在生物医学中起的各种作用至关重要。冷冻电子层析成像（Cryo-ET）与子体积平均（SVA）相结合，目前是唯一允许在纳米分辨率下在其未扰动的本机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大型复合物或有序的组件，并且具有太低而无法揭示分子水平相互作用的分辨率。该技术开发项目的总体目标是设计计算工具，以改善Cryo-Et/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。 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靶标类别，包括低分子量蛋白，目前被认为低于该技术的技术能力。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改善了最新方法，可用于在近原子分辨率下确定小型300kDa酶的结构，这是该领域的重要里程碑。我们的研究之所以创新，是因为它试图克服Cryo-Et中所需的基本技术挑战，以实现这种新兴成像技术的全部潜力。该提案很重要，因为它表明可以使用Cryo-ET可视化具有挑战性的靶标，这表明该技术是研究重要生物分子的最有希望的途径。最终，通过缩小研究样品的策略之间的分辨率差距和在其天然细胞环境中研究蛋白质的技术，我们的方法将允许以空前的细节水平的功能状态可视化蛋白质的功能状态。