Atomic resolution protein structure determination by three-dimensional transferred echo double resonance solid-state nuclear magnetic resonance spectroscopy
Atomic resolution protein structure determination by three-dimensional transferred echo double resonance solid-state nuclear magnetic resonance spectroscopy
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DOI：
10.1063/1.3211103
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发表时间：
2009-09-07
影响因子：
4.4
通讯作者：
Rienstra, Chad M.
中科院分区：
文献类型：
--
作者：
Nieuwkoop, Andrew J.;Wylie, Benjamin J.;Rienstra, Chad M.
我们表明，通过魔角旋转固态核磁共振波谱，可以获得足够数量的定量核间 N-15-C-13 距离，以确定中等大小蛋白质的完整、高分辨率结构。三维 ZF-TEDOR 脉冲序列与免疫球蛋白结合蛋白 G (GB1) β 1 结构域中 C-13 位点的稀疏标记结合使用，如通过使用 1,3-C-13 或 2-C-13-甘油作为 C-13 来源的细菌表达获得的。从二维 N-15-C-13 平面中提取定量偶极轨迹，其中解析了类似于 750 个交叉峰。实验数据符合自旋团簇（每个由一个 C-13 和多个 N-15 组成）的精确理论轨迹，对于大约 350 个位点产生高达 0.1 A 度的定量精度，对于另外 150 个位点优于 0.3 A 度，对于 5-8 A 度范围内的 150 个距离，产生类似于 1.0 A 度的定量精度。与基于各向同性化学位移 (TALOS) 的二面角约束一起，距离约束被纳入模拟退火计算中，以产生高精度结构（主链 RMSD 为 0.25 +/- 0.09 A 度），这也表明与 GB1 最密切相关的晶体结构（2QMT，bbRMSD 0.79 +/- 0.03 A 度）具有极好的一致性。此外，侧链重原子受到很好的限制（0.76 +/- 0.06 A 度总重原子 RMSD）。这些结果首次证明可以测量整个固体蛋白质的定量核间距离，以产生原子分辨率的结构。
We show that quantitative internuclear N-15-C-13 distances can be obtained in sufficient quantity to determine a complete, high-resolution structure of a moderately sized protein by magic-angle spinning solid-state NMR spectroscopy. The three-dimensional ZF-TEDOR pulse sequence is employed in combination with sparse labeling of C-13 sites in the beta 1 domain of the immunoglobulin binding protein G (GB1), as obtained by bacterial expression with 1,3-C-13 or 2-C-13-glycerol as the C-13 source. Quantitative dipolar trajectories are extracted from two-dimensional N-15-C-13 planes, in which similar to 750 cross peaks are resolved. The experimental data are fit to exact theoretical trajectories for spin clusters (consisting of one C-13 and several N-15 each), yielding quantitative precision as good as 0.1 A degrees for similar to 350 sites, better than 0.3 A degrees for another 150, and similar to 1.0 A degrees for 150 distances in the range of 5-8 A degrees. Along with isotropic chemical shift-based (TALOS) dihedral angle restraints, the distance restraints are incorporated into simulated annealing calculations to yield a highly precise structure (backbone RMSD of 0.25 +/- 0.09 A degrees), which also demonstrates excellent agreement with the most closely related crystal structure of GB1 (2QMT, bbRMSD 0.79 +/- 0.03 A degrees). Moreover, side chain heavy atoms are well restrained (0.76 +/- 0.06 A degrees total heavy atom RMSD). These results demonstrate for the first time that quantitative internuclear distances can be measured throughout an entire solid protein to yield an atomic-resolution structure.