Refinement of Macromolecular Assembly Structure using Electron Microscopy

描述（由申请人提供）：细胞的许多关键功能是通过复杂的组装完成的，其结构和构象变化最好通过电子显微镜显示。在样品制备，仪器和图像处理的进步支持可视化在增加分辨率，但通常不足以独立的结构测定。在这里，将开发计算机方法，以充分利用已知的高分辨率组件结构-阐明亚基之间的分子相互作用，组装时的结构变化和功能状态之间的构象转变。已经开发了用于将已知组件结构初始对接到组装密度的方法。在这里，重点是更详细的灵活模型的细化。首先将研究基础，例如适合不同分辨率的模型的粒度和参数化。我们开发的模型交叉验证方法可以客观地解决这些问题。然后将评估立体化学限制，以确定哪种最有效地补充可用的低分辨率数据。这项工作将包括发展的限制连续静电和氢键，并比较原子的限制与histististics，可能会提高收敛在低分辨率。最后，将评估各种优化算法，包括最小二乘法，蒙特卡罗和遗传算法。将制定准则，以选择最适合分辨率范围和实验制度的改进方法。标准将是模型细节的最大化，而不会过度拟合，正如新的交叉验证指标所判断的那样。最好的方法将编入一个软件包分发。它们将分别与Ken Taylor和Joachim Frank合作应用于肌肉蛋白质和核糖体动力学的电子显微镜研究。初步结果表明，潜在的结构细节提取远远超出了标称分辨率限制，使用限制细化。一个目标是探索更合适的参数化和原子细化的限制，提高一般的电子显微镜研究的产品。第二个目标是对生物分子复合物进行更详细的机械理解，超越结构域运动到驱动它们的内部构象变化。

DESCRIPTION (provided by applicant): Many of the cell's critical functions are accomplished by complex assemblies whose structures and conformational changes are best revealed by electron microscopy. Advances in sample preparation, instrumentation and image processing have supported visualization at increasing resolution, though usually insufficient for independent structure determination. Here, computer methods will be developed to take full advantage of known high resolution component structures - to elucidate molecular interactions between subunits, structural changes upon assembly and conformational transitions between functional states. Methods are already developed for the initial docking of known component structures into assembly density. Here, the focus is on refinement of more detailed flexible models. The foundations will be investigated first, such as the granularity and parameterization of models appropriate at different resolutions. Our development of model cross-validation methods allows these issues to be tackled objectively. Then stereochemical restraints will be evaluated to see which most effectively complements the available low resolution data. This work will include development of restraints for continuum electrostatics and hydrogen bonding, and a comparison of atomistic restraints with heuristics that might enhance convergence at low resolution. Finally, a variety of optimization algorithms will be evaluated, including least-squares, Monte Carlo and genetic algorithms. Guidelines will be developed to choose the refinement approaches best suited to the resolution range and experimental regime. The criteria will be maximization of model detail without over-fitting, as judged by the new cross-validation metrics. The best methods will be programmed in a software package to be distributed. They will be applied to electron microscopic studies of muscle protein and ribosome dynamics in collaborations with Ken Taylor and Joachim Frank respectively. Preliminary results demonstrate the potential for extraction of structural detail well beyond the nominal resolution limit, using restrained refinement. One goal is to probe the limits with more appropriate parameterization and restraint of the atomic refinement, enhancing generally the product of electron microscopy studies. A second goal is a more detailed mechanistic understanding of biomolecular complexes, going beyond domain movements to the internal conformational changes that drive them.