REVIEW ARTICLE FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices Eitan Lerner1†*, Anders Barth2†*, Jelle Hendrix3†*, Benjamin Ambrose4, Victoria Birkedal5, Scott C Blanchard6, Richard Bo¨ rner7, Hoi Sung Chung8, Thorben Cordes9, Timothy D Craggs4, Ashok A Deniz10, Jiajia Diao11, Jingyi Fei12, Ruben L Gonzalez13, Irina V Gopich8, Taekjip Ha14, Christian A Hanke2, Gilad Haran15, Nikos S Hatzakis16,17, Sungchul Hohng18, Seok-Cheol Hong19, Thorsten Hugel20, Antonino Ingargiola21, Chirlmin Joo22, Achillefs N Kapanidis23, Harold D Kim24, Ted Laurence25, Nam Ki Lee26, Tae-Hee Lee27, Edward A Lemke28,29, Emmanuel Margeat30, Jens Michaelis31, Xavier Michalet21, Sua Myong32, Daniel Nettels33, Thomas-Otavio Peulen34, Evelyn Ploetz35, Yair Razvag1, Nicole C Robb36, Benjamin Schuler33, Hamid Soleimaninejad37, Chun Tang38, Reza Vafabakhsh39, Don C Lamb35*, Claus AM Seidel2*, Shimon Weiss21,40* 1Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, and The Center for Nanoscience and Nanotechnology, Faculty of Mathematics & Science, The Edmond J. Safra Campus, The Hebrew University of 2 *For correspondence: Jerusalem, Jerusalem, Israel; Lehrstuhl fu¨ r Molekulare Physikalische Chemie, [email protected] (EL); Heinrich-Heine-Universita¨ t, Du¨ sseldorf, Germany; 3Dynamic Bioimaging Lab, [email protected] (AB); Advanced Optical Microscopy Centre and Biomedical Research Institute (BIOMED), [email protected] (JH); Hasselt University, Diepenbeek, Belgium; 4Department of Chemistry, University of [email protected] (DCL); 5 [email protected] (CAMS); Sheffield, Sheffield, United Kingdom; Department of Chemistry and iNANO center, 6 [email protected] (SW) Aarhus University, Aarhus, Denmark; Department of Structural Biology, St. Jude 7 †These authors contributed Children’s Research Hospital, Memphis, United States; Laserinstitut HS Mittweida, 8 equally to this work University of Applied Science Mittweida, Mittweida, Germany; Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Competing interests: The 9 authors declare that no National Institutes of Health, Bethesda, United States; Physical and Synthetic competing interests exist. Biology, Faculty of Biology, Ludwig-Maximilians-Universita¨ t Mu¨ nchen, Planegg- Martinsried, Germany; 10Department of Integrative Structural and Computational Funding: See page 41 Biology, The Scripps Research Institute, La Jolla, United States; 11Department of Received: 29 June 2020 Cancer Biology, University of Cincinnati School of Medicine, Cincinnati, United Accepted: 09 February 2021 12 Published: 29 March 2021 States; Department of Biochemistry and Molecular Biology and The Institute for Biophysical Dynamics, University of Chicago, Chicago, United States; 13Department Reviewing editor: Olga of Chemistry, Columbia University, New York, United States; 14Department of Boudker, Weill Cornell Medicine, Biophysics and Biophysical Chemistry, Department of Biomedical Engineering, United States Johns Hopkins University School of Medicine, Howard Hughes Medical Institute, This is an open-access article, Baltimore, United States; 15Department of Chemical and Biological Physics, free of all copyright, and may be 16 freely reproduced, distributed, Weizmann Institute of Science, Rehovot, Israel; Department of Chemistry & 17 transmitted, modified, built Nanoscience Centre, University of Copenhagen, Copenhagen, Denmark; Denmark upon, or otherwise used by Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and anyone for any lawful purpose. Medical Sciences, University of Copenhagen, Copenhagen, Denmark; 18Department The work is made available under of Physics and Astronomy, and Institute of Applied Physics, Seoul National the Creative Commons CC0 19 public domain dedication. University, Seoul, Republic of Korea; Center for Molecular Spectroscopy and Lerner, Barth, Hendrix, et al. eLife 2021;10:e60416. DOI: https://doi.org/10.7554/eLife.60416 1 of 69 Review Article Biochemistry and Chemical Biology Structural Biology and Molecular Biophysics Dynamics, Institute for Basic Science and Department of Physics, Korea University, Seoul, Republic of Korea; 20Institute of Physical Chemistry and Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany; 21Department of Chemistry and Biochemistry, and Department of Physiology, University of California, Los Angeles, Los Angeles, United States; 22Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, Netherlands; 23Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, United Kingdom; 24School of Physics, Georgia Institute of Technology, Atlanta, United States; 25Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, United States; 26School of Chemistry, Seoul National University, Seoul, Republic of Korea; 27Department of Chemistry, Pennsylvania State University, University Park, United States; 28Departments of Biology and Chemistry, Johannes Gutenberg University, Mainz, Germany; 29Institute of Molecular Biology (IMB), Mainz, Germany; 30Centre de Biologie Structurale (CBS), CNRS, INSERM, Universitie´ de Montpellier, Montpellier, France; 31Institu¨ t of Biophysics, Ulm University, Ulm, Germany; 32Department of Biophysics, Johns Hopkins University, Baltimore, United States; 33Department of Biochemistry and Department of Physics, University of Zurich, Zurich, Switzerland; 34Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, United States; 35Physical Chemistry, Department of Chemistry, Center for Nanoscience (CeNS), Center for Integrated Protein Science Munich (CIPSM) and Nanosystems Initiative Munich (NIM), Ludwig-Maximilians-Universita¨ t, Mu¨ nchen, Germany; 36Warwick Medical School, University of Warwick, Coventry, United Kingdom; 37Biological Optical Microscopy Platform (BOMP), University of Melbourne, Parkville, Australia; 38College of Chemistry and Molecular Engineering, PKU-Tsinghua Center for Life Sciences, Beijing National Laboratory for Molecular Sciences, Peking University, Beijing, China; 39Department of Molecular Biosciences, Northwestern University, Evanston, United States; 40Department of Physiology, CaliforniaNanoSystems Institute, University of California, Los Angeles, Los Angeles, United States Abstract Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever- increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB- Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices. Lerner, Barth, Hendrix, et al. eLife 2021;10:e60416. DOI: https://doi.org/10.7554/eLife.60416 2 of 69 Review Article Biochemistry and Chemical Biology Structural Biology and Molecular Biophysics Introduction Understanding how biomolecules couple structural dynamics with function is at the heart of several disciplines and remains an outstanding goal in biology. Linking conformational states and their tran- sitions to biochemical function requires the ability to precisely resolve the structure and dynamics of a biological system, which is often altered upon ligand binding or influenced by the chemical and physical properties of its environment. The most well-established structural biology tools have pro- vided high-resolution ‘snapshots’ of states in a crystallized or frozen form (e.g., X-ray crystallography and single-particle cryo-electron microscopy, cryoEM) or an ensemble average of all contributing conformations (e.g., nuclear magnetic resonance, NMR; small-angle X-ray scattering, SAXS; small- angle neutron scattering, SANS; double electron-electron resonance, DEER; cross-linking mass spec- trometry, XL-MS; ensemble-FRET). In recent years, further developments have enabled these con- ventional structural tools to detect conformational dynamics and reaction intermediates. For example, NMR techniques (Anthis and Clore, 2015; Clore and Iwahara, 2009; Palmer, 2004; Ravera et al., 2014; Sekhar and Kay, 2019) and electron paramagnetic resonance techniques (Jeschke, 2018; Jeschke, 2012; Krstic´ et al., 2011) have been advanced to study conformational dynamics and capture transient intermediates. Time-resolved crystallographic investigations have been employed to resolve functionally relevant