STRUCTURAL ANALYSIS OF MACROMOLECULAR ASSEMBLY

描述（由申请人提供）：Cryo-EM结合单粒子重建，是Pi实验室首创的一种方法，能够在不同功能状态的天然条件下可视化大分子机器，如转录复合物，伴侣或核糖体。核糖体是一种不对称的结构，其目标是获得10a，这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，感谢NIH的持续资助。软件（SPIDER）的传播使其他团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，在9-13 a的分辨率范围内解释了广泛的功能复合物的密度图，大大提高了我们对因子结合的认识和我们对翻译动力学的理解。在本提案的具体目标#1中设定了一个新的分辨率目标：实现某些已知高度稳定的核糖体复合物的原子（~3A）分辨率。这一目标需要在几个方面同时努力：(i)探索网格制备参数，以获得具有最佳厚度的均匀冰层；（ii）收集的数据量大幅增加（约10倍，产量为1,000,000个“好粒子”），需要提高自动化水平和计算资源；（iii）校正CTF的策略；（iv）角度细化方法，必须通过设计和实施更有效的算法和计算策略来加速和改进；(v)必须探索x射线结构与密度图的拟合和对接方法。Specific Aim #2涉及在真核生物和真细菌中mRNA-tRNA易位的关键步骤（GTP水解）的近原子分辨率的阐明，将通过在Specific Aim #1中开发和实施的技术应用于具有已证实的稳定性的特定复合物来实现。在朝着3a目标前进的道路上，我们可以预期，由于rRNA结构的改进定义，以及蛋白质亚域和结构基序(如A -螺旋和-？-薄片，允许在易位过程中的构象变化和分子相互作用以更高的精度建模和描述。为了实现这些目标，我们与重建算法、核糖体生物化学、x射线晶体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.