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《快与死:小核酶和核糖开关晶体结构快速相位分析指南》

The Quick and the Dead: A Guide to Fast Phasing of Small Ribozyme and Riboswitch Crystal Structures.

作者信息

Jenkins Jermaine L, Wedekind Joseph E

机构信息

Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box 712, Rochester, NY, 14642, USA.

Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, 14642, USA.

出版信息

Methods Mol Biol. 2016;1490:265-80. doi: 10.1007/978-1-4939-6433-8_17.

Abstract

Ribozymes and riboswitches are examples of non-protein-coding (nc)RNA molecules that achieve biological activity by adopting complex three-dimensional folds. Visualization of such molecules at near-atomic resolution can enhance our understanding of how chemical groups are organized spatially, thereby providing novel insight into function. This approach has its challenges, which mainly entail sample crystallization followed by the application of empirical, structure-determination methods that often include experimental "phasing" of X-ray diffraction data. A paucity of high-quality crystals or a low symmetry space group are factors that demand rapid assessment of phasing potential during an ongoing experiment in order to assure a successful outcome. Here we describe the process of evaluating the anomalous signal-to-noise as a prelude to single wavelength or multiwavelength anomalous diffraction (SAD or MAD) phasing. Test cases include an autolytic 62-mer RNA enzyme known as the hairpin ribozyme, and a 33-mer riboswitch that binds the modified guanine metabolite preQ1. The crystals were derivatized with iridium (III) hexammine and osmium (III) pentaammine triflate, respectively. Each data set was then subjected to the XPREP and SHELX programs to assess the anomalous signal-to-noise and to locate the heavy-atom substructure. Subsequent noise filtering was conducted in SHELXE or RESOLVE. The methods described are applicable to the rapid phasing of RNA X-ray diffraction data, and contrast the efficacy of in-house X-rays with those attainable from synchrotron-radiation sources in terms of the potential to plan for and execute an experimental structure determination.

摘要

核酶和核糖开关是非蛋白质编码(nc)RNA分子的实例,它们通过形成复杂的三维折叠来实现生物活性。以近原子分辨率对这类分子进行可视化,可以增强我们对化学基团如何在空间上组织的理解,从而为其功能提供新的见解。这种方法存在挑战,主要包括样品结晶,然后应用经验性的结构测定方法,这些方法通常包括对X射线衍射数据进行实验“相位确定”。高质量晶体的缺乏或低对称空间群是在正在进行的实验中需要快速评估相位潜力以确保成功结果的因素。在这里,我们描述了评估异常信噪比的过程,作为单波长或多波长异常衍射(SAD或MAD)相位确定的前奏。测试案例包括一种称为发夹核酶的自裂解62聚体RNA酶,以及一种结合修饰鸟嘌呤代谢物preQ1的33聚体核糖开关。晶体分别用六氨合铱(III)和三氟甲磺酸五氨合锇(III)进行衍生化。然后对每个数据集进行XPREP和SHELX程序处理,以评估异常信噪比并定位重原子子结构。随后在SHELXE或RESOLVE中进行噪声过滤。所描述的方法适用于RNA X射线衍射数据的快速相位确定,并对比了实验室X射线与同步辐射源在规划和执行实验结构测定潜力方面的功效。

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