STRUCTURAL ANALYSIS OF MACROMOLECULAR ASSEMBLY

描述(申请人提供)：冷冻-EM结合单粒子重建，这是PI的实验室首创的方法，能够在自然条件下以不同的功能状态可视化大分子机器，如转录复合体、伴侣或核糖体。核糖体是一种没有对称性的结构，为了达到10A的目标，推动了我们实验室数据收集和图像处理技术的发展。这一目标已经实现，这要归功于NIH对这笔赠款的持续资助。软件(SPIDER)的传播使其他群体对许多其他生物结构的研究受益。在翻译的研究中，通过X射线结构的拟合解释了在9-13A分辨率范围内的广泛的功能络合物的密度图，极大地促进了我们对因子结合的认识和对翻译动力学的理解。在本提案的具体目标#1中设定了一个新的拆分目标：实现对某些已知高度稳定的核糖体复合体的原子(~3A)拆分。实现这一目标需要在以下几个领域同时努力：(1)探索产生具有最佳厚度的均匀冰层的网格制备参数；(2)收集的数据大幅增加(约10倍，产生1,000,000个“好粒子”)，这需要更高的自动化水平和计算资源；(3)进行CTF校正的战略；(4)角度细化方法，必须通过设计和实施更高效的算法和计算战略来加快和改进这种方法；(5)必须探索将X射线结构拟合和对接密度图的方法。具体目标#2涉及以近原子分辨率阐明在真细菌和真核生物中的mRNA-tRNA转位的关键步骤(GTP水解)，这将通过将在特定目标#1中开发和实施的技术应用于具有已证明稳定性的特定络合物来实现。在朝着3A的目标前进的过程中，我们可以预期，由于对rRNA结构以及蛋白质亚域和结构基序(如α-螺旋和-β-折叠)的定义的改进，将会有新的发现，从而允许以更高的精度模拟和描述移位过程中的构象变化和分子相互作用。为了实现这些目标，我们与重建算法、核糖体生物化学、X射线结晶学和计算建模领域的关键专家合作。

DESCRIPTION (provided by applicant): Cryo-EM combined with single-particle reconstruction, a method pioneered in the Pi's lab, is capable of visualizing macromolecular machines such as transcription complexes, chaperones, or ribosomes under native conditions in different functional states. The goal of attaining 10 A for the ribosome, a structure without symmetry, has driven development of data collection and image processing techniques in our lab. This goal has been achieved, thanks to consistent funding of this grant from NIH. The dissemination of software (SPIDER) has benefited the research of other groups on numerous other biological structures. In the study of translation, density maps for a broad range of functional complexes in the resolution range of 9-13 A, interpreted by fitting of X-ray structures, have greatly advanced our knowledge of factor binding and our understanding of the dynamics of translation. A new resolution goal is set in Specific Aim #1 of the present proposal: the achievement of atomic (~3A) resolution for certain ribosomal complexes known to be highly stable. This goal requires simultaneous efforts in several areas: (i) exploration of grid preparation parameters that lead to uniform layers of ice with optimum thickness; (ii) large increase in data collected (~10-fold, for a yield of 1,000,000 "good particles"), requiring an increased level of automation and computational resources; (iii) strategies for carrying out CTF correction; (iv) angular refinement methods, which must be accelerated and improved through design and implementation of more efficient algorithms and computational strategies; (v) methods for fitting and docking of X-ray structures into density maps must be explored. Specific Aim #2 concerns the elucidation, at near-atomic resolution, of a pivotal step (GTP hydrolysis) in mRNA-tRNA translocation both in eubacteria and eukaryotes, to be achieved by application of the technology developed and implemented in Specific Aim #1 to specific complexes with proven stability. Already along the way toward the goal of 3 A, we can expect that new discoveries will be made due to the improved definition of rRNA structure, as well as protein subdomains and structural motifs such as a-helices and -?-sheets, allowing conformational changes and molecular interactions during translocation to be modeled and described with much higher precision. To achieve these goals, we collaborate with key experts in the areas of reconstruction algorithms, ribosome biochemistry, X-ray crystallography, and computational modeling.