Study of nucleic acid structure by novel NMR methods

开发了一种通过添加稀疏实验数据来精化同源模型的方法。该方法用于确定E.Coli tRNAVal的结构，最初是根据酵母tRNAPhe的X射线结构建模的，但使用实验剩余偶极耦合(RDC)和小角X射线散射(SAXS)数据进行了改进。已经针对SAXS数据开发了一种球面采样算法，该算法不需要球面近似，这对核酸特别重要，因为这种近似不太合适。该算法的显著更高的速度也使其应用于蛋白质。除了SAXS数据外，结构改进还采用了一组稀疏的核磁共振数据，其中包括24个通过PF1噬菌体比对测量的imino N-HN RDC，以及20个通过tRNAVal的磁场依赖比对获得的imino N-HN RDC。精化策略旨在通过确保核糖-磷酸主干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的局部几何结构。局部坐标约束是使用XploR-NIH或CNS软件包中的非晶体对称性(NCS)项来实施的，同时仍然允许相邻片段的适度移动。RDC主要驱动螺旋臂的相对取向，而SAXS约束确保了整体分子形状与实验散射数据相一致。所得结构显示出良好的交叉验证统计数据(PF1 RDC的杰克切割QFree=14%，而起始模型为25%)，并且显示出比在tRNAPhe的X射线结构中观察到的更大的两个螺旋臂之间的角度，这与以前基于核磁共振的tRNAVal模型一致。

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.