Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Frankfurt Institute for Advanced Studies, Goethe University Frankfurt am Main, Max-von-Laue-Str. 9, 60438, Frankfurt am Main, Germany.
J Biomol NMR. 2013 Oct;57(2):193-204. doi: 10.1007/s10858-013-9779-x. Epub 2013 Sep 15.
Protein structure determination by NMR can in principle be speeded up both by reducing the measurement time on the NMR spectrometer and by a more efficient analysis of the spectra. Here we study the reliability of protein structure determination based on a single type of spectra, namely nuclear Overhauser effect spectroscopy (NOESY), using a fully automated procedure for the sequence-specific resonance assignment with the recently introduced FLYA algorithm, followed by combined automated NOE distance restraint assignment and structure calculation with CYANA. This NOESY-FLYA method was applied to eight proteins with 63-160 residues for which resonance assignments and solution structures had previously been determined by the Northeast Structural Genomics Consortium (NESG), and unrefined and refined NOESY data sets have been made available for the Critical Assessment of Automated Structure Determination of Proteins by NMR project. Using only peak lists from three-dimensional (13)C- or (15)N-resolved NOESY spectra as input, the FLYA algorithm yielded for the eight proteins 91-98 % correct backbone and side-chain assignments if manually refined peak lists are used, and 64-96 % correct assignments based on raw peak lists. Subsequent structure calculations with CYANA then produced structures with root-mean-square deviation (RMSD) values to the manually determined reference structures of 0.8-2.0 Å if refined peak lists are used. With raw peak lists, calculations for 4 proteins converged resulting in RMSDs to the reference structure of 0.8-2.8 Å, whereas no convergence was obtained for the four other proteins (two of which did already not converge with the correct manual resonance assignments given as input). These results show that, given high-quality experimental NOESY peak lists, the chemical shift assignments can be uncovered, without any recourse to traditional through-bond type assignment experiments, to an extent that is sufficient for calculating accurate three-dimensional structures.
通过缩短在 NMR 光谱仪上的测量时间以及更有效地分析光谱，可以从原理上加快通过 NMR 确定蛋白质结构的速度。在此，我们研究了基于单一类型光谱（即核 Overhauser 效应光谱（NOESY））确定蛋白质结构的可靠性，使用最近引入的 FLYA 算法进行全自动序列特异性共振分配，然后使用 CYANA 进行自动 NOE 距离约束分配和结构计算。NOESY-FLYA 方法已应用于 8 种具有 63-160 个残基的蛋白质，这些蛋白质的共振分配和溶液结构先前已由东北结构基因组学联盟（NESG）确定，并且为 NMR 项目的蛋白质自动结构确定的关键评估提供了未精制和精制的 NOESY 数据集。仅使用三维（13）C 或（15）N 分辨 NOESY 光谱的峰列表作为输入，如果使用手动精修峰列表，则 FLYA 算法可为这 8 种蛋白质产生 91-98％的正确主链和侧链分配，如果基于原始峰列表，则分配的正确率为 64-96％。然后使用 CYANA 进行后续结构计算，如果使用精制峰列表，则产生的结构与手动确定的参考结构的均方根偏差（RMSD）值为 0.8-2.0 Å。如果使用原始峰列表，则有 4 种蛋白质的计算结果收敛，得到的 RMSD 值与参考结构为 0.8-2.8 Å，而对于另外 4 种蛋白质则未收敛（其中两种蛋白质即使使用正确的输入手动共振分配也无法收敛）。这些结果表明，在具有高质量实验性 NOESY 峰列表的情况下，可以在无需进行传统的基于键的分配实验的情况下，在足以计算准确的三维结构的程度上，揭示化学位移分配。
