Structural Biology of Macromolecular Assemblies

大分子组装体的结构生物学

• 批准号: 9563893
• 负责人: ALASDAIR C. STEVEN
• 金额: $ 59.26万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9563893
• 关键词: Adhesives; Air; Bacteria; Bacteriophages; Binding; Biological; Caliber; Capsid; Carbohydrates; Cell membrane; Classification; Code; Complex; Computer software; Crowding; Cryoelectron Microscopy; Crystallization; Data; Development; Dimensions; Disease; Disulfides; Dose; Electron Microscopy; Electrons; Ensure; Ferritin; Galactose; Glues; Glycolipids; Glycoproteins; Goals; Gram-Negative Bacteria; Head; Heterogeneity; Homeostasis; Human; Image; Image Analysis; In Situ; Intention; Intercellular Junctions; Iron; Lead; Libraries; Lipid Bilayers; Lipids; Longevity; Maps; Membrane Proteins; Modeling; Modernization; Morphologic artifacts; Motion; Mutation; Myxococcus xanthus; N-terminal; Oxidative Stress; Paper; Phase; Photoreceptors; Preparation; Process; Property; Proteins; Publishing; Reporting; Resolution; Retina; Schedule; Scheme; Series; Side; Site; Structure; Structure-Activity Relationship; Universities; Update; Validation; Virus; Visual impairment; Water; Work; Writing; X-Linked Retinoschisis; X-Ray Crystallography; XLRS1 protein; computer program; computerized tools; density; image processing; improved; macromolecular assembly; macromolecule; male; meetings; movie; mutant; particle; reconstruction; scaffold; structural biology

The goal of this project is to elucidate structure-function relationships in macromolecular assemblies. During FY17, our studies focused on retinoschisin (RS1), a junctional protein in the human retina; encapsulin, a bacterial nanocompartment that sequesters iron; and computational tools used in image analysis. 1) RS1 is a protein required to maintain the structural and functional integrity of the retina. Mutations in RS1 lead to early vision impairment in young males, a condition termed X-linked retinoschisis (XLRS). From earlier work, RS1 was thought to form an octamer, with each subunit comprising a discoidin domain (DS) and a small N-terminal domain (RS1 domain). We used cryo-EM to determine the structure of RS1 at 0.4 nm resolution, finding that the complex consists, in fact, of two apposed octameric rings. The RS1 domains occupy the centers of the rings, but are less clearly defined, suggesting mobility. We combined the cryo-EM density map with crystal structures of other discoidin domains to create a high-resolution model of the double octamer. This model is consistent with intramolecular and intermolecular disulfides previously reported. The interfaces internal to and between rings accommodate residues implicated in XLRS, indicating the importance of correct assembly of the 16-meric complex to obtain a correctly constituted junction. We published a paper reporting this analysis in May 2016 (G. Tolun et al., Proc Natl Acad Sci USA 113:5287-92). Since then, the project has been extended as follows. From the known sites of disease-causing RS1 mutants, adhesive functionality appears to be associated with the spikes located at the periphery of the rings. RS1 is thought to interact with cell membranes by binding to lipid head-groups, via membrane proteins and/or carbohydrate moieties on glycolipids or glycoproteins. In particular, RS1 has been shown to bind to galactose. Accordingly, we performed cryoEM on RS1 with galactose bound. To our surprise, we found that the double rings form long branched chains, constituting a 2D network. In these chains, the molecules mostly present side-views, suggesting that they may interact with the air-water interface through their spikes. The interface is thought to be a mimic for lipid bilayers. Within chains there are spike-spike interactions between neighboring molecules. The spikes are also involved in branching interactions, where they mostly interact with the tops of the rings on another molecule. The ability of RS1 to form such a network suggests that in situ it may be forming a 3D scaffold between photoreceptors to glue them together. A paper reporting this phase of the project is in preparation. 2) Encapsulin is a virus capsid-like nanocompartment that sequesters iron, thereby protecting bacteria from oxidative stress. In earlier work reported in FY15, we characterized the structure of encapsulin isolated from the Gram-negative bacterium Myxococcus xanthus. This particle has a protein shell assembled from 180 copies of EncA protein, and smaller amounts of three internal proteins (EncB; EncC; EncD). Using cryo-EM, we showed that EncA assembles into an icosahedral capsid 32 nm in diameter with a triangulation number of T=3. Our analysis showed that EncA has the fold first observed in bacteriophage HK97 capsid. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, but with a massively greater capacity (30,000 Fe atoms vs. 3,000 in ferritin). In FY17, our main thrust has been an attempt to seek high resolution cryo-EM data on particles of the purified internal protein ClpB. The data obtained to date are encouraging in the sense that EncB makes quite large particles, enhancing the feasibility of this approach, but progress has been hampered by overly crowded grids and eventual heterogeneity of the particles. 3) Development of image processing software for three-dimensional electron microscopy. Bsoft is a comprehensive suite of computer programs for image processing of cryo-EM images and cryo-ET data that is maintained, disseminated, and further developed in the LSBR by B. Heymann. In FY17, an updated and upgraded version of Bsoft (Bsoft 2.0.0) was released. In it, the code structure has been modified to eliminate legacy libraries and to introduce a more general compilation scheme. The intention is to develop along more modern standards with coding in C++ to ensure better stability and longevity. The single particle analysis (SPA) capabilities have been expanded to allow better 2D analysis and classification. Specifically, the handling of dose-fractionated movies (motion correction) has been improved; the processing of tomographic tilt series can now be done through the Bshow interface without the need to write command lines. This includes estimation of the contrast transfer function parameters and correcting for it during reconstruction. In the last few years, advances in cryo-EM and image processing have made possible density maps at resolutions comparable to those achieved by X-ray crystallography. This development has raised the question of whether any particular work-flows or processing strategies achieve the best results and/or whether artifacts may be introduced in some circumstances. To this end, B. Heymann is participating in the Map Challenge project intended to assess processing and map validation (http://challenges.emdatabank.org/?q=2015_map_challenge). A meeting to discuss the results and conclusions is scheduled at Stanford University, Oct 5-8, 2017. The conclusions will be published.

该项目的目标是阐明大分子组装体中的结构-功能关系。 2017 财年，我们的研究重点是视网膜裂素 (RS1)，这是人类视网膜中的一种连接蛋白；封装蛋白，一种可以螯合铁的细菌纳米室；以及图像分析中使用的计算工具。 1) RS1是维持视网膜结构和功能完整性所需的蛋白质。 RS1 突变会导致年轻男性早期视力障碍，这种情况称为 X 连锁视网膜劈裂症 (XLRS)。从早期的工作来看，RS1被认为形成一个八聚体，每个亚基包含一个盘状蛋白结构域（DS）和一个小的N端结构域（RS1结构域）。我们使用冷冻电镜以 0.4 nm 分辨率确定 RS1 的结构，发现该复合物实际上由两个并置的八聚环组成。 RS1 域占据环的中心，但定义不太明确，表明具有移动性。我们将冷冻电镜密度图与其他盘状蛋白结构域的晶体结构相结合，创建了双八聚体的高分辨率模型。该模型与先前报道的分子内和分子间二硫化物一致。环内部和环之间的界面容纳 XLRS 中涉及的残基，表明正确组装 16 聚体复合物以获得正确构成的连接的重要性。我们于 2016 年 5 月发表了一篇论文报告了这项分析（G. Tolun 等人，Proc Natl Acad Sci USA 113:5287-92）。 此后，该项目已扩展如下。 从已知的致病 RS1 突变体位点来看，粘附功能似乎与位于环外围的尖峰有关。 RS1被认为通过膜蛋白和/或糖脂或糖蛋白上的碳水化合物部分与脂质头基结合而与细胞膜相互作用。特别是，RS1 已被证明可以与半乳糖结合。因此，我们对结合了半乳糖的 RS1 进行了冷冻电镜检查。令我们惊讶的是，我们发现双环形成长支链，构成二维网络。在这些链中，分子大多呈现侧视图，表明它们可能通过尖峰与空气-水界面相互作用。该界面被认为是脂质双层的模拟。在链内，相邻分子之间存在尖峰-尖峰相互作用。尖峰还参与分支相互作用，其中它们主要与另一个分子上的环顶部相互作用。 RS1 形成这种网络的能力表明，它可能在光感受器之间原位形成 3D 支架，将它们粘合在一起。一份报告该项目这一阶段的文件正在准备中。 2) 封装蛋白是一种病毒衣壳状纳米室，可以隔离铁，从而保护细菌免受氧化应激。在 2015 财年报道的早期工作中，我们表征了从革兰氏阴性细菌黄粘球菌中分离出的封装蛋白的结构。该颗粒具有由 180 个 EncA 蛋白拷贝和少量的三种内部蛋白（EncB；EncC；EncD）组装而成的蛋白壳。使用冷冻电镜，我们发现 EncA 组装成直径 32 nm 的二十面体衣壳，三角剖分数 T=3。我们的分析表明，EncA 具有首次在噬菌体 HK97 衣壳中观察到的折叠。天然纳米室具有致密的富铁核心。从功能上讲，它们类似于铁蛋白，但容量大得多（铁蛋白为 30,000 个铁原子，而铁原子为 30,000 个）。 2017 财年，我们的主要目标是尝试寻找纯化内部蛋白 ClpB 颗粒的高分辨率冷冻电镜数据。迄今为止获得的数据令人鼓舞，因为 EncB 制造了相当大的颗粒，增强了这种方法的可行性，但由于网格过于拥挤和颗粒最终的异质性，进展受到阻碍。 3）三维电子显微镜图像处理软件开发。 Bsoft 是一套全面的计算机程序，用于冷冻电镜图像和冷冻电子断层扫描数据的图像处理，由 B. Heymann 在 LSBR 中维护、传播和进一步开发。 2017财年，发布了Bsoft的更新升级版本（Bsoft 2.0.0）。其中，代码结构已被修改，以消除遗留库并引入更通用的编译方案。目的是使用 C++ 编码按照更现代的标准进行开发，以确保更好的稳定性和寿命。单粒子分析 (SPA) 功能已得到扩展，可实现更好的 2D 分析和分类。具体来说，改进了剂量分割电影（运动校正）的处理；现在可以通过 Bshow 界面完成层析倾斜系列的处理，而无需编写命令行。这包括估计对比度传递函数参数并在重建期间对其进行校正。 在过去的几年中，冷冻电镜和图像处理的进步使得密度图的分辨率与 X 射线晶体学所达到的分辨率相当。这一发展提出了这样的问题：任何特定的工作流程或处理策略是否能够实现最佳结果和/或在某些情况下是否可能引入工件。为此，B. Heymann 正在参与地图挑战项目，旨在评估处理和地图验证 (http://challenges.emdatabank.org/?q=2015_map_challenge)。定于 2017 年 10 月 5 日至 8 日在斯坦福大学召开会议讨论结果和结论。结论将予以公布。