Tang Yuefeng, Schneider William M, Shen Yang, Raman Srivatsan, Inouye Masayori, Baker David, Roth Monica J, Montelione Gaetano T
Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Northeast Structural Genomics Consortium, Rutgers University, Piscataway, NJ 08854, USA.
J Struct Funct Genomics. 2010 Dec;11(4):223-32. doi: 10.1007/s10969-010-9095-6. Epub 2010 Aug 24.
Determination of high-quality small protein structures by nuclear magnetic resonance (NMR) methods generally requires acquisition and analysis of an extensive set of structural constraints. The process generally demands extensive backbone and sidechain resonance assignments, and weeks or even months of data collection and interpretation. Here we demonstrate rapid and high-quality protein NMR structure generation using CS-Rosetta with a perdeuterated protein sample made at a significantly reduced cost using new bacterial culture condensation methods. Our strategy provides the basis for a high-throughput approach for routine, rapid, high-quality structure determination of small proteins. As an example, we demonstrate the determination of a high-quality 3D structure of a small 8 kDa protein, E. coli cold shock protein A (CspA), using <4 days of data collection and fully automated data analysis methods together with CS-Rosetta. The resulting CspA structure is highly converged and in excellent agreement with the published crystal structure, with a backbone RMSD value of 0.5 Å, an all atom RMSD value of 1.2 Å to the crystal structure for well-defined regions, and RMSD value of 1.1 Å to crystal structure for core, non-solvent exposed sidechain atoms. Cross validation of the structure with (15)N- and (13)C-edited NOESY data obtained with a perdeuterated (15)N, (13)C-enriched (13)CH(3) methyl protonated CspA sample confirms that essentially all of these independently-interpreted NOE-based constraints are already satisfied in each of the 10 CS-Rosetta structures. By these criteria, the CS-Rosetta structure generated by fully automated analysis of data for a perdeuterated sample provides an accurate structure of CspA. This represents a general approach for rapid, automated structure determination of small proteins by NMR.
通过核磁共振（NMR）方法测定高质量的小蛋白质结构通常需要获取和分析大量的结构约束条件。这个过程一般需要大量的主链和侧链共振归属，以及数周甚至数月的数据收集和解读。在这里，我们展示了使用CS-Rosetta快速生成高质量蛋白质NMR结构的方法，该方法使用了通过新的细菌培养浓缩方法制备的、成本显著降低的全氘代蛋白质样品。我们的策略为小蛋白质的常规、快速、高质量结构测定的高通量方法提供了基础。例如，我们展示了使用<4天的数据收集以及与CS-Rosetta一起的全自动数据分析方法，测定了一个8 kDa小蛋白质——大肠杆菌冷休克蛋白A（CspA）的高质量三维结构。所得的CspA结构高度收敛，与已发表的晶体结构非常吻合，主链均方根偏差（RMSD）值为0.5 Å，对于明确界定的区域，与晶体结构的全原子RMSD值为1.2 Å，对于核心、非溶剂暴露的侧链原子，与晶体结构的RMSD值为1.1 Å。用全氘代（15）N、（13）C富集的（13）CH（3）甲基质子化CspA样品获得的（15）N和（13）C编辑的NOESY数据对该结构进行交叉验证，证实了在10个CS-Rosetta结构中的每一个中，基本上所有这些基于NOE的独立解释的约束条件都已得到满足。根据这些标准，通过对全氘代样品数据进行全自动分析生成的CS-Rosetta结构提供了CspA的准确结构。这代表了一种通过NMR快速、自动测定小蛋白质结构的通用方法。
