Rapid protein global fold determination using ultrasparse sampling, high-dynamic range artifact suppression, and time-shared NOESY.
Rapid protein global fold determination using ultrasparse sampling, high-dynamic range artifact suppression, and time-shared NOESY.
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DOI：
10.1021/ja307445y
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发表时间：
2012-11-14
影响因子：
15
通讯作者：
Zhou P
中科院分区：
文献类型：
--
作者：
Coggins BE;Werner-Allen JW;Yan A;Zhou P
在通过NMR进行的大蛋白质结构研究中，全局折叠确定在提供对目标拓扑结构的第一次观察和减少完全质子化样品的NOESY光谱中的分配模糊性方面起着越来越重要的作用。在这项工作中，我们展示了使用超解析采样，一种新的数据处理算法，和一个4-D的时间共享NOESY实验（1）收集所有NOE在2 H/13 C/15 N标记的蛋白质样品与选择性质子化酰胺和ILV甲基基团在高分辨率在只有四天，（2）计算全局折叠从这个数据使用全自动共振分配。新算法SCRUB采用CLEAN方法进行迭代伪影消除，但应用了额外的迭代级别，允许将真实的信号与噪声区分开来，并允许消除由真实的信号产生的几乎所有伪影。在模拟中，奈奎斯特采样所需数据的1.2%，SCRUB实现了超过10000：1的动态范围（比CLEAN高250倍的伪影抑制），并完全定量再现信号强度，体积和线形。应用于4-D分时NOESY数据，SCRUB处理可显著降低强对角信号的混叠噪声，从而能够识别强度比对角信号小100倍的弱NOE串扰。几乎所有预期的质子间距离在5 μ m以下的峰值都被观测到了。使用CYANA的自动分配协议，其中未分配的4-D分时NOESY峰列表产生准确且收敛良好的全局折叠集合，而3-D峰列表要么不能收敛，要么产生明显不太准确的折叠，通过23 kDa和29 kDa测试蛋白的结构计算证明了该方法的实际益处。这里提出的方法成功地与一个数量级少的采样比处理稀疏4-D数据的替代方法所需的。
In structural studies of large proteins by NMR, global fold determination plays an increasingly important role in providing a first look at a target’s topology and reducing assignment ambiguity in NOESY spectra of fully-protonated samples. In this work, we demonstrate the use of ultrasparse sampling, a new data processing algorithm, and a 4-D time-shared NOESY experiment (1) to collect all NOEs in 2H/13C/15N-labeled protein samples with selectively-protonated amide and ILV methyl groups at high resolution in only four days, and (2) to calculate global folds from this data using fully automated resonance assignment. The new algorithm, SCRUB, incorporates the CLEAN method for iterative artifact removal, but applies an additional level of iteration, permitting real signals to be distinguished from noise and allowing nearly all artifacts generated by real signals to be eliminated. In simulations with 1.2% of the data required by Nyquist sampling, SCRUB achieves a dynamic range over 10000:1 (250× better artifact suppression than CLEAN) and completely quantitative reproduction of signal intensities, volumes, and lineshapes. Applied to 4-D time-shared NOESY data, SCRUB processing dramatically reduces aliasing noise from strong diagonal signals, enabling the identification of weak NOE crosspeaks with intensities 100× less than diagonal signals. Nearly all of the expected peaks for interproton distances under 5 Å were observed. The practical benefit of this method is demonstrated with structure calculations for 23 kDa and 29 kDa test proteins using the automated assignment protocol of CYANA, in which unassigned 4-D time-shared NOESY peak lists produce accurate and well-converged global fold ensembles, whereas 3-D peak lists either fail to converge or produce significantly less accurate folds. The approach presented here succeeds with an order of magnitude less sampling than required by alternative methods for processing sparse 4-D data.
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