Breaking Barriers in Structural Biology: Novel CryoEM Methods and Applications

打破结构生物学的障碍：新颖的冷冻电镜方法和应用

• 批准号: 9349372
• 负责人: Dmitry Lyumkis
• 金额: $ 48.5万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-25 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9349372
• 关键词: 3-Dimensional; Address; Adhesions; Automation; Biochemical; Biological; Biology; Bypass; Cell Differentiation process; Chimeric Proteins; Classification; Cloning; Collaborations; Complex; Computer software; Critical Pathways; Cryoelectron Microscopy; Crystallization; Data; Development; Drug Design; Drug Targeting; Escherichia coli; Explosion; Faculty; Family; Foundations; Future; Genes; Goals; Human; IKK alpha; Immunity; In Vitro; Individual; Inflammation; Inflammatory Response; Institutes; Knock-out; Laboratories; Libraries; Macromolecular Complexes; Mediating; Methodology; Methods; Mission; Modeling; Molecular; Molecular Analysis; Molecular Models; Nature; Noise; Pathway interactions; Play; Preparation; Proteins; Public Health; Regulation; Research; Research Infrastructure; Research Personnel; Resolution; Ribosomal Proteins; Ribosomes; Roentgen Rays; Role; Signal Pathway; Signal Transduction; Specimen; Structural Biologist; Structure; System; Techniques; Technology; Transcriptional Regulation; Translating; United States National Institutes of Health; Virus; Work; Yeasts; base; density; drug discovery; improved; in vivo; insight; instrumentation; interest; macromolecular assembly; macromolecule; model building; new technology; novel; particle; protein complex; public health relevance; structural biology; targeted treatment; tool; transcription factor

DESCRIPTION (provided by applicant): Single-particle cryo-electron microscopy (cryoEM) has witnessed an explosion of activity and interest in recent years, as certain biological structures that were previously extremely challenging to solve have become much more tractable using the technology. Some structures, like icosahedral viruses and ribosomes, are now being solved to near-atomic resolution on a routine basis. The capabilities imply that atomic-level structural information is potentially achievable for many long sought-after protein targets, thus opening doors for exciting discoveries in structural biology. However, the inherently low signal-to-noise ratio of the acquired data makes certain targets extremely challenging to study using the technique, and the resolution will be limited to large domains, at best. In this application, one of the major challenges in single-particle cryoEM will be addressed with the development of a methodology that would enable routine structure solution of small (<100 kDa) macromolecules and macromolecular complexes. In parallel, the existing technological infrastructure, together with methodological improvements, will be applied to an outstanding problem in biology - the cryoEM structure of the human IKK complex, a central regulator of NF-κB based transcription regulation and a key target for drug design. Despite previous efforts using X-ray based techniques, the structure of IKK, and a rational structure-based model of its activation, remains elusive. The utilization of cryoEM to solve the structure of IKK will bypass the difficulties associated with specimen crystallization, while building on the inherent advantages of single-particle techniques, specifically in their ability to characterize dynamic and heterogeneous macromolecular assemblies. This work will provide groundwork for future functional analyses that will be performed in collaboration with research groups in the immediate vicinity of the laboratory and is expected to a broad impact on drug design efforts aimed at the IKK complex.

描述（由适用提供）：近年来，单粒子冷冻电子显微镜（Cryoem）见证了活动和兴趣的爆炸，因为使用该技术，某些以前对解决方案极为挑战的生物学结构变得更加易于解决。某些结构，例如二十面体病毒和核糖体，现在正在常规上解决近原子分辨率。该功能表明，对于许多长期扫描的蛋白质靶标而言，原子水平的结构信息可能是可以接受的，从而为结构生物学的令人兴奋的发现打开了大门。但是，获得的数据固有的较低信噪比使某些目标挑战使用该技术极为挑战，并且该分辨率最多仅限于大型域。在此应用中，将通过开发一种方法来解决单粒子冷冻的主要挑战之一，该方法可以实现小（<100 kDa）大分子和大分子复合物的常规结构解决方案。同时，现有的技术基础设施以及方法论改进将应用于生物学的杰出问题 - 人IKK复合物的冷冻结构，它是基于NF -κB的转录调节的中心调节器，也是药物设计的关键目标。尽管以前使用基于X射线的技术进行了努力，但IKK的结构以及基于合理的结构激活模型仍然是弹性的。冷冻解决IKK结构的利用将绕过与样品结晶相关的困难，同时建立在单粒子技术的继承优势的同时，特别是其表征动态和异构的大分子分子组件的能力。这项工作将为未来的功能分析提供基础，这些功能分析将与实验室附近的研究小组合作进行，并有望对针对IKK综合体的药物设计工作产生广泛的影响。