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

基本信息

批准号：
9002750
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
项目类别：
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/9002750
关键词：
3-Dimensional Address Adhesions Automation Biochemical Biological Biology Bypass Cell Differentiation process Chimeric Proteins Classification 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 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 Protein Dynamics Proteins Public Health Regulation Research Research Infrastructure Research Personnel Resolution Ribosomal Proteins Ribosomes Roentgen Rays Role Signal Pathway Signal Transduction Solutions Specimen Structural Biologist Structure System Techniques Technology Transcriptional Regulation Translating United States National Institutes of Health Virus Work Yeasts base density drug discovery expression cloning improved in vivo insight instrumentation interest macromolecular assembly macromolecule model building molecular modeling new technology novel particle protein complex public health relevance structural biology targeted treatment tool transcription factor

项目摘要

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（由申请人提供）：近年来，单粒子冷冻电子显微镜（cryoEM）的活动和兴趣激增，因为以前极难解决的某些生物结构使用该技术变得更加容易处理。像二十面体病毒和核糖体一样，现在正在常规基础上以近原子分辨率解决这些问题，这意味着原子级结构信息对于许多长期追捧的人来说是可能实现的。蛋白质靶点，从而为结构生物学中令人兴奋的发现打开了大门。所采集数据的低信噪比使得使用该技术研究某些目标极具挑战性，并且分辨率充其量也仅限于大域，在此应用中，单粒子冷冻电镜的主要挑战之一将是。通过开发一种能够对小（<100 kDa）大分子和大分子复合物进行常规结构解决的方法来解决这一问题。同时，现有的技术基础设施以及方法的改进将应用于解决生物学中的一个突出问题——冷冻电镜人类 IKK 复合物的结构，基于 NF-κB 的转录调控的中心调节剂和药物设计的关键目标，尽管之前使用基于 X 射线的技术进行了努力，但 IKK 的结构及其激活的基于合理结构的模型。利用冷冻电镜来解决 IKK 的结构将绕过与样本结晶相关的困难，同时建立在单粒子技术的固有优势上，特别是在表征动态和特征的能力方面。这项工作将为未来的功能分析奠定基础，这些分析将与实验室附近的研究小组合作进行，预计将对 IKK 复合物的药物设计工作产生广泛影响。