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Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study

机译:单分子FRET测量的精度和准确性 - 一种多实验室基准研究

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Single-molecule Forster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between +/- 0.02 and +/- 0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.
机译:单分子福斯特共振能量转移(SMFRet)越来越多地用于确定体外和体内生物分子的距离,结构和动力学。然而,目前缺乏全面的协议和FRET标准,以确保荧光效率测量的再现性和准确性。在这里,我们报告了对比例盲目研究的结果,其中20实验室确定了几种染料标记的DNA双链体的FRET效率(e)。我们使用统一的简单方法,我们获得了S.D的荧光效率。 +/- 0.02和+/- 0.05之间。我们建议将FRET效率转化为准确距离的实验和计算程序,并讨论实验和建模中的潜在不确定性。我们对基于强度的SMFRet测量和统一校正程序的再现性的定量评估以及朝向距离网络验证的重要步骤,最终目的是通过SMFRET的混合方法实现生物分子系统的可靠结构模型。

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