Structural Analysis of Macromolecular Assemblies

 描述(由申请人提供)：本提案寻求继续支持通过低温电子显微镜(CRYO-EM)和单粒子重建来探索活跃在蛋白质合成中的核糖体的结构和功能。蛋白质的生物合成是生命最基本的过程之一，但尽管在解决核糖体结构方面做了开创性的工作，但这个过程还没有被很好地理解。CRYO-EM是唯一适合在接近自然条件下捕获处于功能状态的分子的仪器。在这个实验室中，过去在真细菌和真核细胞翻译中关于这一过程的动力学已经有了几个开创性的发现；其中包括在mRNA-tRNA转位过程中的棘轮状运动，以及在氨酰-tRNA选择过程中氨基酰-tRNA进入核糖体时的大弹簧样变形。随着最近分辨率的提高，核糖体复合体的结构可以以接近原子的分辨率可视化。这项研究计划的重点将是真核(寄生虫和哺乳动物)核糖体的结构和动力学。拟议研究的目标有三个：(1)解决四种已知引起人类衰弱疾病的真核寄生虫核糖体的结构和功能动力学；(2)通过选择起始复合体的冷冻-EM可视化，研究哺乳动物真核翻译启动的过程；(3) 研究某些病毒(EMCV、脊髓灰质炎)侵占哺乳动物宿主的翻译器官以制造组装新病毒副本所需的蛋白质的过程。样本将从几个专门从事真核翻译的合作实验室获得。当密度图以足够的分辨率获得时，将通过灵活的拟合进行分析，并在现有结构、动力学、单分子FRET和生化数据的丰富背景下进行解释。分辨率等于或高于3.0A的密度图将用于原子结构的从头计算建模。

 DESCRIPTION (provided by applicant): This proposal seeks continued support for exploration of structure and function of the ribosome actively engaged in protein synthesis, by cryo-electron microscopy (cryo-EM) and single-particle reconstruction. Protein biosynthesis is one of the most fundamental processes of life, but despite seminal work in solving the structure of the ribosome, this process is not well understood. Cryo-EM is uniquely suited to capture the molecule in functional states under close to native conditions. In this lab, several seminal discoveries regarding the dynamics of this process in both eubacterial and eukaryotic translation have been made in the past; among these the ratchet-like motion during mRNA-tRNA translocation and the large spring-like deformation of the aminoacyl-tRNA as it enters the ribosome during the aminoacyl-tRNA selection process. With the recent increase in resolution, structures of ribosomal complexes can be visualized with close to atomic resolutions. Special focus of this research program will be the structure and dynamics of the eukaryotic (parasites and mammalian) ribosomes. The aims of the proposed studies are threefold: (1) to solve the structures, and characterize the functional dynamics, of ribosomes from four eukaryotic parasites known to cause debilitating human diseases; (2) to study the process of mammalian eukaryotic translation initiation by cryo-EM visualization of selected initiation complexes; (3) to study the process by which certain viruses (EMCV, polio) usurp the translational apparatus of the mammalian host to make protein required for assembly of new copies of the virus. Samples will be obtained from several collaborating labs that are specialized in eukaryotic translation. Density maps when obtained at sufficient resolution will be analyzed by flexible fitting and interpreted in the rich context of existing structural, kinetics, single-molecule FRET, and biochemical data. Density maps with resolutions equal or better than 3.0 A will be used for ab initio modeling of the atomic structure.