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.

这个项目的目标是阐明大分子组装中的结构-功能关系。在2017财年，我们的研究重点是视黄蛋白(RS1)，这是人类视网膜中的一种连接蛋白；包裹素，一种隔离铁的细菌纳米隔膜；以及用于图像分析的计算工具。 1)RS1是维持视网膜结构和功能完整性所必需的蛋白质。RS1基因突变会导致年轻男性早期视力障碍，这种情况被称为X-连锁视网膜劈裂(XLRS)。根据早期的工作，RS1被认为形成一个八聚体，每个亚基包括一个盘状结构域(DS)和一个小的N-末端结构域(RS1结构域)。我们用低温电子显微镜在0.4 nm分辨率下确定了RS1的结构，发现该络合物实际上是由两个相对的八聚体环组成的。RS1结构域占据环的中心，但定义不太清楚，表明可迁移性。我们将冷冻EM密度图与其他盘状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。该模型与已报道的分子内和分子间二硫键相一致。环内和环之间的界面容纳了XLRS中涉及的残基，这表明正确组装16-Meric复合体对于获得正确构成的连接非常重要。我们在2016年5月发表了一篇报告这一分析的论文(G.Tolun等人，Proc Natl Acad Sci USA 113：5287-92)。从那时起，该项目得到了如下扩展。 从已知的致病RS1突变体的位置来看，粘连功能似乎与位于环外围的尖刺有关。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细胞膜结合，从而与细胞膜相互作用。特别是，RS1已被证明与半乳糖结合。因此，我们对半乳糖结合的RS1进行了冷冻EM。令我们惊讶的是，我们发现双环形成了长长的支链，构成了一个2D网络。在这些链中，分子大多呈现侧视，表明它们可能通过它们的尖峰与空气-水界面相互作用。这种界面被认为是类脂双层的模仿物。在链中，相邻分子之间存在尖峰-尖峰相互作用。这些尖峰还参与了分支相互作用，它们主要与另一个分子上的环的顶部相互作用。RS1形成这种网络的能力表明，它可能在原位形成了光感受器之间的3D支架，将它们粘合在一起。一份报告该项目这一阶段的论文正在编写中。 2)包囊是一种类似病毒衣壳的纳米隔膜，可以隔离铁，从而保护细菌免受氧化应激的伤害。在2015财年报道的早期工作中，我们表征了从革兰氏阴性杆菌黄色粘球菌分离出的囊膜的结构。这种颗粒有一个由180个拷贝的eNCA蛋白和少量的三种内部蛋白(EncB；EncC；EncD)组装而成的蛋白质外壳。利用冷冻-EM技术，我们发现eNCA组装成一个直径为32 nm的二十面体衣壳，三角数目为T=3。我们的分析表明，eNCA具有在噬菌体HK97衣壳中首次观察到的折叠。天然纳米隔间具有致密的富含铁的核心。在功能上，它们类似于铁蛋白，但容量要大得多(30,000个铁原子与3,000个铁蛋白)。在2017财年，我们的主要任务是尝试寻找关于纯化的内部蛋白ClpB颗粒的高分辨率冷冻-EM数据。到目前为止获得的数据令人鼓舞，因为EncB制造了相当大的颗粒，增强了这一方法的可行性，但进展受到格子过于拥挤和颗粒最终不均匀的阻碍。 3)三维电子显微镜图像处理软件的开发。Bsoft是一套全面的计算机程序，用于冷冻EM图像和冷冻数据的图像处理，由B.Heymann在LSBR中维护、传播和进一步开发。在2017财年，发布了Bsoft的更新和升级版本(Bsoft 2.0.0)。在它中，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。其目的是通过使用C++编写代码来开发更现代的标准，以确保更好的稳定性和寿命。单粒子分析(SPA)功能已经扩展，可以进行更好的2D分析和分类。具体地说，改进了对剂量分割电影(运动校正)的处理；断层扫描倾斜序列的处理现在可以通过Bshow界面完成，而不需要编写命令行。这包括对比度传递函数参数的估计和在重建期间对其进行校正。 在过去的几年里，低温电子显微镜和图像处理的进步使得密度图的分辨率可以与X射线结晶学的密度图相媲美。这一发展提出了一个问题，即是否有任何特定的工作流程或处理策略达到最佳结果，以及/或者是否可能在某些情况下引入构件。为此，B.海曼正在参与地图挑战项目，该项目旨在评估处理和地图验证(http://challenges.emdatabank.org/?q=2015_map_challenge).2017年10月5日至8日，将在斯坦福大学举行会议，讨论结果和结论。结论将会公布。