Study of nucleic acid structure by novel NMR methods

通过新型 NMR 方法研究核酸结构

• 批准号: 7734035
• 负责人: Ad - Bax
• 金额: $ 30.49万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734035
• 关键词: Agreement; Algorithms; Bacteriophage Pf1; Base Pairing; Behavior; Class; Compatible; Complement; Computer software; Coupling; Crystallization; Data; Ensure; Exhibits; Homology Modeling; Measures; Messenger RNA; Methods; Modeling; Movement; Nucleic Acids; Phenylalanine-Specific tRNA; Procedures; Proteins; Relative (related person); Residual state; Roentgen Rays; Sampling; Solutions; Speed; Structure; Transfer RNA; Upper arm; Validation; Valine-Specific tRNA; Vertebral column; Yeasts; base; data structure; magnetic field; molecular shape; novel; nucleic acid structure; restraint; ribose phosphate; statistics

A procedure has been developed for refinement of homology models by addition of sparse experimental data. The method is demonstrated for determining the structure of E.Coli tRNAVal, originally modeled after the X-ray structure of yeast tRNAPhe, but refined using experimental residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. A spherical sampling algorithm has been developed for refinement against SAXS data that does not require a globbic approximation, which is particularly important for nucleic acids where such approximations are less appropriate. Substantially higher speed of the algorithm also makes its application favorable for proteins. In addition to the SAXS data, the structure refinement employed a sparse set of NMR data consisting of 24 imino N-HN RDCs measured with Pf1 phage alignment, and 20 imino N-HN RDCs obtained from magnetic field dependent alignment of tRNAVal. The refinement strategy aims to largely retain the local geometry of the 58% identical tRNAPhe by ensuring that the atomic coordinates for short, overlapping segments of the ribose-phosphate backbone and the conserved base pairs remain close to those of the starting model. Local coordinate restraints are enforced using the non-crystallographic symmetry (NCS) term in the XPLOR-NIH or CNS software package, while still permitting modest movements of adjacent segments. The RDCs mainly drive the relative orientation of the helical arms, whereas the SAXS restraints ensure an overall molecular shape compatible with experimental scattering data. The resulting structure exhibits good cross-validation statistics (jack-knifed Qfree = 14% for the Pf1 RDCs, compared to 25% for the starting model) and exhibits a larger angle between the two helical arms than observed in the X-ray structure of tRNAPhe, in agreement with previous NMR-based tRNAVal models.

一个程序已经开发了细化的同源性模型，增加了稀疏的实验数据。该方法被证明用于确定大肠杆菌tRNAVal的结构，最初是根据酵母tRNAPhe的X射线结构建模的，但使用实验残留偶极耦合（RDC）和小角X射线散射（SAXS）数据进行了改进。已经开发了球形采样算法用于针对SAXS数据的细化，其不需要全局近似，这对于其中这种近似不太合适的核酸特别重要。该算法的速度大大提高，也使其适用于蛋白质。 除了SAXS数据外，结构精修还采用了稀疏的NMR数据集，该数据集由Pf 1噬菌体比对测量的24个亚氨基N-HN RDC和从tRNAVal的磁场依赖性比对获得的20个亚氨基N-HN RDC组成。改进策略旨在通过确保核糖磷酸骨架和保守碱基对的短重叠片段的原子坐标保持接近起始模型的原子坐标，在很大程度上保留58%相同的tRNAPhe的局部几何形状。使用XPLOR-NIH或CNS软件包中的非晶体学对称性（NCS）项强制执行局部坐标约束，同时仍允许相邻节段的适度移动。 RDC主要驱动螺旋臂的相对取向，而SAXS约束确保与实验散射数据兼容的整体分子形状。得到的结构表现出良好的交叉验证统计（折刀Qfree = 14%的Pf 1 RDC，相比之下，25%的起始模型），并表现出更大的角度之间的两个螺旋臂比观察到的X射线结构的tRNAPhe，与以前的NMR为基础的tRNAVal模型。