Study of nucleic acid structure and dynamics by novel NMR methods

本文提出了一种通过添加稀疏实验数据来细化合理起始模型的方法。该方法最初是根据酵母tRNAPhe的x射线结构建模的，但使用实验残余偶极耦合（RDC）和小角x射线散射（SAXS）数据进行了改进。已经开发了一种球面采样算法，用于针对不需要全局近似的SAXS数据进行细化，这对于这种近似不太合适的核酸尤其重要。较高的算法速度也使其有利于蛋白质的应用。除了SAXS数据外，结构精化还使用了一组稀疏的NMR数据，其中包括Pf1噬菌体定位测量的24个极小N-HN rdc，以及tRNAVal磁场依赖定位获得的20个极小N-HN rdc。改进策略的目的是通过确保核糖-磷酸盐主链的短重叠片段和保守碱基对的原子坐标保持与初始模型的原子坐标接近，从而在很大程度上保留58%相同tRNAPhe的局部几何形状。使用XPLOR-NIH或CNS软件包中的非晶体对称（NCS）项强制执行局部坐标约束，同时仍然允许相邻段的适度运动。rdc主要驱动螺旋臂的相对方向，而SAXS约束确保整体分子形状与实验散射数据兼容。由此产生的结构具有良好的交叉验证统计（Pf1 rdc的jack-刀Qfree = 14%，而初始模型为25%），并且与tRNAPhe的x射线结构相比，两个螺旋臂之间的角度更大，与之前基于核磁共振的tRNAVal模型一致。包含残余化学位移各向异性（CSA）作为结构约束的补充来源取决于对最小15N共振的静态CSA张量的准确了解。利用tRNA_Val的结构作为参考点，确定了A和G核苷酸的CSA张量的大小和取向。了解CSA值也是确定核碱基动态性质的关键，这是目前正在研究的课题。

A procedure has been developed for refinement of plausible starting 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. Inclusion of residual chemical shift anisotropy (CSA) as a complementary source of structural restraint hinges on accurate knowledge of the static CSA tensor for imino 15N resonances. The magnitude and orientation of the CSA tensors has been determined for both A and G nucleotides, using the structure of tRNA_Val as a reference point. Knowledge of the CSA values is also key to determining the dynamic properties of the nucleobases, a subject currently under investigation.