Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.
Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.
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DOI：
10.1038/s41592-023-01807-0
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发表时间：
2023-04
期刊：
影响因子：
48
通讯作者：
Cordes, Thorben
中科院分区：
文献类型：
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作者：
Agam, Ganesh;Gebhardt, Christian;Popara, Milana;Maechtel, Rebecca;Folz, Julian;Ambrose, Benjamin;Chamachi, Neharika;Chung, Sang Yoon;Craggs, Timothy D.;de Boer, Marijn;Grohmann, Dina;Ha, Taekjip;Hartmann, Andreas;Hendrix, Jelle;Hirschfeld, Verena;Huebner, Christian G.;Hugel, Thorsten;Kammerer, Dominik;Kang, Hyun-Seo;Kapanidis, Achillefs N.;Krainer, Georg;Kramm, Kevin;Lemke, Edward A.;Lerner, Eitan;Margeat, Emmanuel;Martens, Kirsten;Michaelis, Jens;Mitra, Jaba;Munoz, Gabriel G. Moya;Quast, Robert B.;Robb, Nicole C.;Sattler, Michael;Schlierf, Michael;Schneider, Jonathan;Schroeder, Tim;Sefer, Anna;Tan, Piau Siong;Thurn, Johann;Tinnefeld, Philip;van Noort, John;Weiss, Shimon;Wendler, Nicolas;Zijlstra, Niels;Barth, Anders;Seidel, Claus A. M.;Lamb, Don C.;Cordes, Thorben
单分子福斯特共振能量转移 (smFRET) 实验可以研究体外和体内的生物分子结构和动力学。我们进行了一项涉及 19 个实验室的国际盲法研究，以评估蛋白质 FRET 实验在测量的 FRET 效率直方图、距离的确定以及结构动力学的检测和量化方面的不确定性。使用两种具有不同构象变化和动力学的蛋白质系统，我们获得了FRET效率的不确定度≤0.06，对应于≤2 Å的染料间距离精度和≤5 Å的准确度。我们进一步讨论了检测该距离范围内波动的限制以及如何识别染料扰动。我们的工作证明了 smFRET 实验能够同时测量距离并避免对现实蛋白质系统的构象动力学进行平均，突显了其在扩展综合结构生物学工具箱中的重要性。一项国际盲法研究证实，动态蛋白质的 smFRET 测量在仪器、分析程序和时间尺度上具有高度可重复性，进一步凸显了 smFRET 在动态结构生物学中的前景。
Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology. An international blind study confirms that smFRET measurements on dynamic proteins are highly reproducible across instruments, analysis procedures and timescales, further highlighting the promise of smFRET for dynamic structural biology.
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影响因子：
16.8
作者：
Gouridis, Giorgos;Schuurman-Wolters, Gea K.;Poolman, Bert
通讯作者：
Poolman, Bert
DOI：
10.1002/cphc.201100897
发表时间：
2012-03
期刊：
Chemphyschem : a European journal of chemical physics and physical chemistry
影响因子：
--
作者：
Felekyan S;Kalinin S;Sanabria H;Valeri A;Seidel CA
通讯作者：
Seidel CA
影响因子：
16.8
作者：
Diez, M;Zimmermann, B;Gräber, P
通讯作者：
Gräber, P
影响因子：
7.7
作者：
de Boer, Marijn;Gouridis, Giorgos;Cordes, Thorben
通讯作者：
Cordes, Thorben
影响因子：
16.8
作者：
通讯作者：
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