Author: Mirabela Rusu, MEng, Ms (2011)
Primary Advisor: Jack W. Smith, MD, PhD
Committee Members: M. Sriram Iyengar, PhD; Todd R. Johnson, PhD; Robert W. Vogler, DSN, MEd
德克萨斯大学生物医学信息学学院的博士学位论文。
抽象的：
基本的生物过程受多种生物分子系统之间有组织的动态相互作用控制。因此，形成了复合物以实现生物学功能并在过程完成后分解。此类过程的例子包括通过核糖体将信使RNA转化为蛋白质，通过伴侣蛋白折叠蛋白或在宿主细胞中的病毒进入。通过表征可以启用的分子机制来理解这些基本过程，这将允许（更好的）疗法和药物设计。这种分子机制可以揭示出对这些过程核心的生物分子组件的结构阐明。可以应用各种实验技术来研究生物分子组件的分子结构。高分辨率技术（例如X射线晶体学）可能会解决系统的原子结构，但通常被限制在降低的质量和尺寸的生物分子上。特别是，X射线晶体学要求样品形成一个三维（3D）晶体晶格，在技术上是在技术上，即使不是不可能的，尤其是对于大型动态系统。通常，这些技术解决了组装中不同成分组成部分的结构，但是在研究整个系统时会遇到di文化。另一方面，成像技术，例如冷冻电子显微镜（Cryo-EM），能够在近本地环境中描绘大型系统，而无需形成晶体。 The structures solved by cryo-EM cover a wide range of resolutions, from very low level of detail where only the overall shape of the system is visible, to high-resolution that approach, but not yet reach, atomic level of detail.
In this dissertation, several modeling methods are introduced to either integrate cryoEM datasets with structural data from X-ray crystallography, or to directly interpret the cryo-EM reconstruction. Such computational techniques were developed with the goal of creating an atomic model for the cryo-EM data. The low-resolution reconstructions lack the level of detail to permit a direct atomic interpretation, i.e. one can not reliably locate the atoms or amino-acid residues within the structure obtained by cryo-EM. Thereby one needs to consider additional information, for example, structural data from other sources such as X-ray crystallography, in order to enable such a high-resolution interpretation. Modeling techniques are thus developed to integrate the structural data from the dierent biophysical sources, examples including the work described in the manuscript I and II of this dissertation. At intermediate and high-resolution, cryo-EM reconstructions depict consistent 3D folds such as tubular features which in general correspond to alpha-helices. Such features can be annotated and later on used to build the atomic model of the system, see manuscript III as alternative.
提出了三个手稿的博士学位dissertation, each introducing a computational technique that facilitates the interpretation of cryo-EM reconstructions. The rst manuscript is an application paper that describes a heuristics to generate the atomic model for the protein envelope of the Rift Valley fever virus. The second manuscript introduces the evolutionary tabu search strategies to enable the integration of multiple component atomic structures with the cryo-EM map of their assembly. Finally, the third manuscript develops further the latter technique and apply it to annotate consistent 3D patterns in intermediate-resolution cryo-EM reconstructions.
第一个手稿标题为“裂谷发烧病毒的装配模型”，在《分子生物学杂志》上发表。Freiberg博士和合作者先前以27Å分辨率解决了Rift Valley Fever病毒的冷冻EM结构。这种重建显示了病毒包膜的整体形状，但细节的降低水平阻止了直接的原子解释。高分辨率结构尚未用于整个病毒，也不适用于形成其包膜的两种不同成分糖蛋白。但是，可以根据原子分辨率可用的类似结构为这些糖蛋白生成同源模型。手稿介绍了基于信封的低分辨率冷冻EM图和两个糖蛋白的同源模型，鉴定整个病毒包膜的原子模型所需的步骤。从详尽的搜索结果开始放置两个糖蛋白的结果开始，该模型是通过运行多个多体性重新元素到层次结构生成模型的模型来构建模型的。生成的原子模型得到有关病毒生物学的先验知识的支持，并包含有关系统分子结构的有价值信息。它为寻求揭示病毒所涉及的不同过程的进一步研究提供了基础，例如组装或融合。
第二个手稿最近发表在the of Journal of Structural Biology (doi:10.1016/j.jsb.2009.12.028) under the title Evolutionary tabu search strategies for the simultaneous registration of multiple atomic structures in cryo-EM reconstructions. This manuscript introduces the evolutionary tabu search strategies applied to enable a multi-body registration. This technique is a hybrid approach that combines a genetic algorithm with a tabu search strategy to promote the proper exploration of the high-dimensional search space. Similar to the Rift Valley fever virus, it is common that the structure of a large multi-component assembly is available at low-resolution from cryo-EM, while high-resolution structures are solved for the dierent components but lack for the entire system. Evolutionary tabu search strategies enable the building of an atomic model for the entire system by considering simultaneously the dierent components. Such registration indirectly introduces spatial constrains as all components need to be placed within the assembly, enabling the proper docked in the low-resolution map of the entire assembly. Along with the method description, the manuscript covers the validation, presenting the bene t of the technique in both synthetic and experimental test cases. Such approach successfully docked multiple components up to resolutions of 40Å.
The third manuscript is entitled Evolutionary Bidirectional Expansion for the Annotation of Alpha Helices in Electron Cryo-Microscopy Reconstructions and was submitted for publication in the Journal of Structural Biology. The modeling approach described in this manuscript applies the evolutionary tabu search strategies in combination with the bidirectional expansion to annotate secondary structure elements in intermediate resolution cryo-EM reconstructions. In particular, secondary structure elements such as alpha helices show consistent patterns in cryo-EM data, and are visible as rod-like patterns of high density. The evolutionary tabu search strategy is applied to identify the placement of the dierent alpha helices, while the bidirectional expansion characterizes their length and curvature. The manuscript presents the validation of the approach at resolutions ranging between 6 and 14Å, a level of detail where alpha helices are visible. Up to resolution of 12 Å, the method measures sensitivities between 70-100% as estimated in experimental test cases, i.e. 70-100% of the alpha-helices were correctly predicted in an automatic manner in the experimental data.
该博士学位论文中介绍的三个手稿涵盖了用于集成和解释冷冻EM重建的不同计算方法。该方法是在分子建模软件雕塑家（http://sculptor.biomachina.org）中开发的，可用于对Cryo-EM数据多分辨率建模感兴趣的科学社区。这项工作涵盖了各种分辨率，涵盖了低分辨率的多体性和注册，并在高分辨率下对一致模式进行注释。这种方法对于冷冻EM数据的建模至关重要，并且可以应用于遇到类似空间问题的其他领域，例如医学成像。