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.

该项目的目的是阐明大分子组件中的结构 - 功能关系。在2017财年期间，我们的研究集中于人视网膜中的连接蛋白视网膜感染素（RS1）。封装，一种细菌纳米室，可隔离铁；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的突变导致年轻男性的早期视力障碍，这种疾病称为X连锁视网膜（XLRS）。从较早的工作中，RS1被认为形成了一个八聚体，每个亚基都包含一个盘状结构域（DS）和一个小的N末端结构域（RS1域）。我们使用冷冻EM在0.4 nm分辨率下确定RS1的结构，发现该复合物实际上是由两个附有八接环组成的。 RS1域占据了环的中心，但明确的定义较不明显，表明流动性。我们将冷冻EM密度图与其他盘状蛋白结构域的晶体结构相结合，以创建双重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。环内部和环之间的接口可容纳与XLR有关的残留物，表明正确组装16分钟复合物以获得正确组成的连接的重要性。我们发表了一篇论文，报道了2016年5月的分析（G. Tolun等，Proc Natl Acad Sci USA 113：5287-92）。 从那时起，该项目已被延长如下。 从已知的引起疾病的RS1突变体的位点，粘合功能似乎与位于环外围的尖峰有关。 RS1被认为通过与脂质头组，通过膜蛋白和/或糖脂蛋白或糖蛋白上的碳水化合物部分结合，与细胞膜相互作用。特别是，RS1已显示与半乳糖结合。因此，我们用半乳糖结合在RS1上进行了冷冻。令我们惊讶的是，我们发现双环形成了长支链，构成了2D网络。在这些链中，分子主要是侧视图，表明它们可以通过尖峰与空气水接口相互作用。该界面被认为是脂质双层的模仿。在链中，相邻分子之间存在尖峰尖峰相互作用。这些尖峰还参与分支相互作用，在分支相互作用中，它们主要与另一个分子上的环的顶部相互作用。 RS1形成这样的网络的能力表明，原位可能在光感受器之间形成3D支架将它们粘合在一起。报告该项目的阶段的论文正在准备。 2）封装是一种类似衣壳的纳米室，可隔离铁，从而保护细菌免受氧化应激。在2015财年报告的早期工作中，我们表征了从革兰氏阴性细菌xanthus中分离出的封装蛋白的结构。该粒子的蛋白壳从180份的Enca蛋白质中组装而成，较小的三种内部蛋白质（ENCB； ENCC； ENCD）。使用Cryo-EM，我们表明ENCA在直径32 nm的二十面体帽中组装，三角剖分数为t = 3。我们的分析表明，ENCA在噬菌体HK97衣壳中首先观察到折叠。天然纳米室具有密集的铁核。从功能上讲，它们类似于铁蛋白，但容量较大（30,000 fe原子与铁蛋白中的3,000）。 在2017财年，我们的主要推力是试图在纯化的内部蛋白质CLPB的颗粒上寻求高分辨率的冷冻EM数据。迄今为止，迄今为止获得的数据令人鼓舞，因为ENCB的粒子是很大的颗粒，增强了这种方法的可行性，但是进步受到过度拥挤的网格和最终的异质性的阻碍。 3）开发用于三维电子显微镜的图像处理软件。 BSOFT是一套全面的计算机程序套件，用于用于处理，B. Heymann在LSBR中维护，传播和进一步开发的冷冻EM图像和冷冻数据的图像处理。在2017财年，发布了更新和升级的BSOFT（BSOFT 2.0.0）的版本。在其中，已修改了代码结构以消除旧版库并引入更通用的汇编方案。目的是通过在C ++中进行编码沿更现代的标准发展，以确保稳定性和寿命改善。单个粒子分析（SPA）功能已扩展，以提供更好的2D分析和分类。具体而言，处理剂量的电影（运动校正）的处理已得到改善。现在可以通过Bshow接口进行层析成像倾斜系列的处理，而无需编写命令行。这包括估计对比度传输函数参数并在重建过程中对其进行校正。 在过去的几年中，冷冻EM和图像处理的进步使在分辨率上的密度图可能与X射线晶体学达到的分辨率相当。这一发展提出了一个问题，即在某些情况下是否可能引入任何特定的工作流或处理策略是否取得最佳结果和/或是否可能引入工件。为此，B。Heymann参加了旨在评估处理和地图验证的地图挑战项目（http://challenges.emdatabank.org/?q=2015_map_challenge）。讨论结果和结论的会议定于2017年10月5日至8日在斯坦福大学举行。结论将发布。