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2. Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Author

Agam, Ganesh, Gebhardt, Christian, Popara, Milana, Mächtel, 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, Hübner, 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, Moya Muñoz, Gabriel G, Quast, Robert B, Robb, Nicole C, Sattler, Michael, Schlierf, Michael, Schneider, Jonathan, Schröder, Tim, Sefer, Anna, Tan, Piau Siong, Thurn, Johann, Tinnefeld, Philip, van Noort, John, Weiss, Shimon, Wendler, Nicolas, Zijlstra, Niels, Barth, Anders, Seidel, Claus AM, Lamb, Don C, and Cordes, Thorben

Subjects
Biochemistry and Cell Biology, Biological Sciences, Affordable and Clean Energy, Fluorescence Resonance Energy Transfer, Reproducibility of Results, Proteins, Molecular Conformation, Laboratories, Technology, Medical and Health Sciences, Developmental Biology, Biological sciences
Abstract

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.

Published
2023

5. The FRET-based structural dynamics challenge -- community contributions to consistent and open science practices

Author

Lerner, Eitan, Ambrose, Benjamin, Barth, Anders, Birkedal, Victoria, Blanchard, Scott C., Borner, Richard, Cordes, Thorben, Craggs, Timothy D., Ha, Taekjip, Haran, Gilad, Hugel, Thorsten, Ingargiola, Antonino, Kapanidis, Achillefs, Lamb, Don C., Laurence, Ted, Lee, Nam ki, Lemke, Edward A., Margeat, Emmanuel, Michaelis, Jens, Michalet, Xavier, Nettels, Daniel, Peulen, Thomas-Otavio, Schuler, Benjamin, Seidel, Claus A. M., So-leimaninejad, Hamid, and Weiss, Shimon

Subjects
Quantitative Biology - Biomolecules, Physics - Biological Physics
Abstract

Single-molecule F\"{o}rster resonance energy transfer (smFRET) has become a mainstream technique for probing biomolecular structural dynamics. The rapid and wide adoption of the technique by an ever-increasing number of groups has generated many improvements and variations in the technique itself, in methods for sample preparation and characterization, in analysis of the data from such experiments, and in analysis codes and algorithms. Recently, several labs that employ smFRET have joined forces to try to bring the smFRET community together in adopting a consensus on how to perform experiments and analyze results for achieving quantitative structural information. These recent efforts include multi-lab blind-tests to assess the accuracy and precision of smFRET between different labs using different procedures, the formal assembly of the FRET community and development of smFRET procedures to be considered for entries in the wwPDB. Here we delve into the different approaches and viewpoints in the field. This position paper describes the current "state-of-the field", points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and a list of resources that are openly available. To make further progress, we strongly encourage 'open science' practices. We hope that this position paper will provide a roadmap for newcomers to the field, as well as a reference for seasoned practitioners.

Published
2020
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6. FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Author

Lerner, Eitan, Barth, Anders, Hendrix, Jelle, Ambrose, Benjamin, Birkedal, Victoria, Blanchard, Scott C, Börner, Richard, Sung Chung, Hoi, Cordes, Thorben, Craggs, Timothy D, Deniz, Ashok A, Diao, Jiajie, Fei, Jingyi, Gonzalez, Ruben L, Gopich, Irina V, Ha, Taekjip, Hanke, Christian A, Haran, Gilad, Hatzakis, Nikos S, Hohng, Sungchul, Hong, Seok-Cheol, Hugel, Thorsten, Ingargiola, Antonino, Joo, Chirlmin, Kapanidis, Achillefs N, Kim, Harold D, Laurence, Ted, Lee, Nam Ki, Lee, Tae-Hee, Lemke, Edward A, Margeat, Emmanuel, Michaelis, Jens, Michalet, Xavier, Myong, Sua, Nettels, Daniel, Peulen, Thomas-Otavio, Ploetz, Evelyn, Razvag, Yair, Robb, Nicole C, Schuler, Benjamin, Soleimaninejad, Hamid, Tang, Chun, Vafabakhsh, Reza, Lamb, Don C, Seidel, Claus Am, and Weiss, Shimon

Subjects
Fluorescence Resonance Energy Transfer, Molecular Biology, Single Molecule Imaging, FRET, biochemistry, biomolecules, chemical biology, community, conformation, dynamics, molecular biophysics, single-molecule, structural biology, Bioengineering, Generic health relevance, Biochemistry and Cell Biology
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.

Published
2021

7. PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins

Author

Lazar, Tamas, Martínez-Pérez, Elizabeth, Quaglia, Federica, Hatos, András, Chemes, Lucía B, Iserte, Javier A, Méndez, Nicolás A, Garrone, Nicolás A, Saldaño, Tadeo E, Marchetti, Julia, Rueda, Ana Julia Velez, Bernadó, Pau, Blackledge, Martin, Cordeiro, Tiago N, Fagerberg, Eric, Forman-Kay, Julie D, Fornasari, Maria S, Gibson, Toby J, Gomes, Gregory-Neal W, Gradinaru, Claudiu C, Head-Gordon, Teresa, Jensen, Malene Ringkjøbing, Lemke, Edward A, Longhi, Sonia, Marino-Buslje, Cristina, Minervini, Giovanni, Mittag, Tanja, Monzon, Alexander Miguel, Pappu, Rohit V, Parisi, Gustavo, Ricard-Blum, Sylvie, Ruff, Kiersten M, Salladini, Edoardo, Skepö, Marie, Svergun, Dmitri, Vallet, Sylvain D, Varadi, Mihaly, Tompa, Peter, Tosatto, Silvio CE, and Piovesan, Damiano

Subjects
Biological Sciences, Bioinformatics and Computational Biology, Generic health relevance, Databases, Protein, Humans, Intrinsically Disordered Proteins, Search Engine, Tumor Suppressor Protein p53, Environmental Sciences, Information and Computing Sciences, Developmental Biology, Biological sciences, Chemical sciences, Environmental sciences
Abstract

The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.

Published
2021

9. Precision and accuracy of single-molecule FRET measurements - a worldwide benchmark study

Author

Hellenkamp, Björn, Schmid, Sonja, Doroshenko, Olga, Opanasyuk, Oleg, Kühnemuth, Ralf, Adariani, Soheila Rezaei, Barth, Anders, Birkedal, Victoria, Bowen, Mark E., Chen, Hongtao, Cordes, Thorben, Eilert, Tobias, Fijen, Carel, Götz, Markus, Gouridis, Giorgos, Gratton, Enrico, Ha, Taekjip, Hanke, Christian A., Hartmann, Andreas, Hendrix, Jelle, Hildebrandt, Lasse L., Hohlbein, Johannes, Hübner, Christian G., Kallis, Eleni, Kapanidis, Achillefs N., Kim, Jae-Yeol, Krainer, Georg, Lamb, Don C., Lee, Nam Ki, Lemke, Edward A., Levesque, Brié, Levitus, Marcia, McCann, James J., Naredi-Rainer, Nikolaus, Nettels, Daniel, Ngo, Thuy, Qiu, Ruoyi, Röcker, Carlheinz, Sanabria, Hugo, Schlierf, Michael, Schuler, Benjamin, Seidel, Henning, Streit, Lisa, Tinnefeld, Philip, Tyagi, Swati, Vandenberk, Niels, Weninger, Keith R., Wünsch, Bettina, Yanez-Orozco, Inna S., Michaelis, Jens, Seidel, Claus A. M., Craggs, Timothy D., and Hugel, Thorsten

Subjects
Quantitative Biology - Quantitative Methods, Physics - Biological Physics
Abstract

Single-molecule F\"orster 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 ensuring both the reproducibility and accuracy of measuring FRET efficiencies are currently lacking. Here we report the results of a worldwide, comparative, blind study, in which 20 labs determined the FRET efficiencies of several dye-labeled DNA duplexes. Using a unified and straightforward method, we show that FRET efficiencies can be obtained with a standard deviation between ${\Delta}$E = +-0.02 and +-0.05. We further suggest an experimental and computational procedure for converting FRET efficiencies into accurate distances. We discuss potential uncertainties in the experiment and the modelling. Our extensive quantitative assessment of intensity-based smFRET measurements and correction procedures serve as an essential step towards validation of distance networks with the ultimate aim to archive reliable structural models of biomolecular systems obtained by smFRET-based hybrid methods., Comment: 27 pages, 5 figures, supplementary material, corrections: p20 last sentence; eqn. 13 & 14

Published
2017
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10. Publisher Correction: Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study

Author

Hellenkamp, Björn, Schmid, Sonja, Doroshenko, Olga, Opanasyuk, Oleg, Kühnemuth, Ralf, Adariani, Soheila Rezaei, Ambrose, Benjamin, Aznauryan, Mikayel, Barth, Anders, Birkedal, Victoria, Bowen, Mark E, Chen, Hongtao, Cordes, Thorben, Eilert, Tobias, Fijen, Carel, Gebhardt, Christian, Götz, Markus, Gouridis, Giorgos, Gratton, Enrico, Ha, Taekjip, Hao, Pengyu, Hanke, Christian A, Hartmann, Andreas, Hendrix, Jelle, Hildebrandt, Lasse L, Hirschfeld, Verena, Hohlbein, Johannes, Hua, Boyang, Hübner, Christian G, Kallis, Eleni, Kapanidis, Achillefs N, Kim, Jae-Yeol, Krainer, Georg, Lamb, Don C, Lee, Nam Ki, Lemke, Edward A, Levesque, Brié, Levitus, Marcia, McCann, James J, Naredi-Rainer, Nikolaus, Nettels, Daniel, Ngo, Thuy, Qiu, Ruoyi, Robb, Nicole C, Röcker, Carlheinz, Sanabria, Hugo, Schlierf, Michael, Schröder, Tim, Schuler, Benjamin, Seidel, Henning, Streit, Lisa, Thurn, Johann, Tinnefeld, Philip, Tyagi, Swati, Vandenberk, Niels, Vera, Andrés Manuel, Weninger, Keith R, Wünsch, Bettina, Yanez-Orozco, Inna S, Michaelis, Jens, Seidel, Claus AM, Craggs, Timothy D, and Hugel, Thorsten

Subjects
Biological Sciences, Good Health and Well Being, Technology, Medical and Health Sciences, Developmental Biology, Biological sciences
Abstract

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Published
2018

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

Author

Hellenkamp, Björn, Schmid, Sonja, Doroshenko, Olga, Opanasyuk, Oleg, Kühnemuth, Ralf, Rezaei Adariani, Soheila, Ambrose, Benjamin, Aznauryan, Mikayel, Barth, Anders, Birkedal, Victoria, Bowen, Mark E, Chen, Hongtao, Cordes, Thorben, Eilert, Tobias, Fijen, Carel, Gebhardt, Christian, Götz, Markus, Gouridis, Giorgos, Gratton, Enrico, Ha, Taekjip, Hao, Pengyu, Hanke, Christian A, Hartmann, Andreas, Hendrix, Jelle, Hildebrandt, Lasse L, Hirschfeld, Verena, Hohlbein, Johannes, Hua, Boyang, Hübner, Christian G, Kallis, Eleni, Kapanidis, Achillefs N, Kim, Jae-Yeol, Krainer, Georg, Lamb, Don C, Lee, Nam Ki, Lemke, Edward A, Levesque, Brié, Levitus, Marcia, McCann, James J, Naredi-Rainer, Nikolaus, Nettels, Daniel, Ngo, Thuy, Qiu, Ruoyi, Robb, Nicole C, Röcker, Carlheinz, Sanabria, Hugo, Schlierf, Michael, Schröder, Tim, Schuler, Benjamin, Seidel, Henning, Streit, Lisa, Thurn, Johann, Tinnefeld, Philip, Tyagi, Swati, Vandenberk, Niels, Vera, Andrés Manuel, Weninger, Keith R, Wünsch, Bettina, Yanez-Orozco, Inna S, Michaelis, Jens, Seidel, Claus AM, Craggs, Timothy D, and Hugel, Thorsten

Subjects
Bioengineering, Affordable and Clean Energy, Fluorescence Resonance Energy Transfer, Laboratories, Reproducibility of Results, Biological Sciences, Technology, Medical and Health Sciences, Developmental Biology
Abstract

Single-molecule Förster 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.

Published
2018

12. Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET

Author

Lu, Maolin, Ma, Xiaochu, Castillo-Menendez, Luis R., Gorman, Jason, Alsahafi, Nirmin, Ermel, Utz, Terry, Daniel S., Chambers, Michael, Peng, Dongjun, Zhang, Baoshan, Zhou, Tongqing, Reichard, Nick, Wang, Kevin, Grover, Jonathan R., Carman, Brennan P., Gardner, Matthew R., Nikić-Spiegel, Ivana, Sugawara, Akihiro, Arthos, James, Lemke, Edward A., Smith, III, Amos B., Farzan, Michael, Abrams, Cameron, Munro, James B., McDermott, Adrian B., Finzi, Andrés, Kwong, Peter D., Blanchard, Scott C., Sodroski, Joseph G., and Mothes, Walther

Published
2019
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13. Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing

Author

Gambin, Yann, VanDelinder, Virginia, Ferreon, Allan Chris M, Lemke, Edward A, Groisman, Alex, and Deniz, Ashok A

Subjects
Biological Sciences, Biotechnology, Bioengineering, Fluorescence Resonance Energy Transfer, Humans, Kinetics, Microfluidic Analytical Techniques, Protein Binding, Protein Folding, alpha-Synuclein, Technology, Medical and Health Sciences, Developmental Biology, Biological sciences
Abstract

We combined rapid microfluidic mixing with single-molecule fluorescence resonance energy transfer to study the folding kinetics of the intrinsically disordered human protein α-synuclein. The time-resolution of 0.2 ms revealed initial collapse of the unfolded protein induced by binding with lipid mimics and subsequent rapid formation of transient structures in the encounter complex. The method also enabled analysis of rapid dissociation and unfolding of weakly bound complexes triggered by massive dilution.

Published
2011

16. Unboxing the treasure chest: tinkering proteins beyond the natural repertoire

Author

Jann, Cosimo and Lemke, Edward A.

Abstract

Proteins are the molecular machineries of life, and engineering them in order to probe and tailor biological function has long remained pure imagination. Cracking the genetic code, the blueprint of proteins, gave the spark to turn precise protein engineering into reality, and even to explore new designer genetic codes. Organisms in all kingdoms of life evolved a highly similar genetic code, which defines how genetic information is translated into proteins. Only a small set of 20 so-called canonical amino acids are used by ribosomes to build proteins. Is the genetic code in this form key to all lives, no matter where in the universe it evolved? Already on Earth we find two powerful exceptions. Selenocysteine represents the 21st canonical amino acid and can even be found in some human proteins. Pyrrolysine is termed the 22nd proteogenic amino acid and has been identified, e.g., in archaeal organisms that grow in anaerobic environments, such as the sludge of a lake or the paunch of a cow. So if one could expand the genetic code even further, proteins could be generated consisting partially or even entirely of artificial amino acid polymers, giving rise to a large diversity of novel natural and unnatural functions. In fact, this future is already present, with genetic code expansion representing a powerful technique which allows to equip proteins with hundreds of non-canonical amino acids, including ones that do not exist in nature. This opens a treasure chest of entirely new protein structures and functionalities. Despite the great potential for biotechnology and medicine, installing a new genetic code, especially in human cells, still bears major challenges. Our laboratory, among others, strives to develop new strategies to expand the genetic code inside of living cells and, at the same time, maintain their native genetic code. Come and join us for this brief journey through the milestones of protein engineering, up to the latest breakthroughs in encoding non-natural chemical properties, and you may get as excited as we are about where protein engineering is heading in the upcoming years.

Published
2023
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18. In situ structural analysis of the human nuclear pore complex

Author

von Appen, Alexander, Kosinski, Jan, Sparks, Lenore, Ori, Alessandro, DiGuilio, Amanda L., Vollmer, Benjamin, Mackmull, Marie-Therese, Banterle, Niccolo, Parca, Luca, Kastritis, Panagiotis, Buczak, Katarzyna, Mosalaganti, Shyamal, Hagen, Wim, Andres-Pons, Amparo, Lemke, Edward A., Bork, Peer, Antonin, Wolfram, Glavy, Joseph S., Bui, Khanh Huy, and Beck, Martin

Subjects
Cytoplasm, Eukaryotes, Cell interaction, Environmental issues, Science and technology, Zoology and wildlife conservation
Abstract

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of [...]

Published
2015

28. Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy

Author

Werther, Philipp, primary, Yserentant, Klaus, additional, Braun, Felix, additional, Grußmayer, Kristin, additional, Navikas, Vytautas, additional, Yu, Miao, additional, Zhang, Zhibin, additional, Ziegler, Michael J., additional, Mayer, Christoph, additional, Gralak, Antoni J., additional, Busch, Marvin, additional, Chi, Weijie, additional, Rominger, Frank, additional, Radenovic, Aleksandra, additional, Liu, Xiaogang, additional, Lemke, Edward A., additional, Buckup, Tiago, additional, Herten, Dirk-Peter, additional, and Wombacher, Richard, additional

Published
2021
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30. FRET-based dynamic structural biology:Challenges, perspectives and an appeal for open-science practices

Author

Lerner, Eitan, Barth, Anders, Hendrix, Jelle, Ambrose, Benjamin, Birkedal, Victoria, Blanchard, Scott C., Börner, Richard, Chung, Hoi Sung, Cordes, Thorben, Craggs, Timothy D., Deniz, Ashok A., Diao, Jiajia, Fei, Jingyi, Gonzalez, Ruben L., Gopich, Irina V., Ha, Taekjip, Hanke, Christian A., Haran, Gilad, Hatzakis, Nikos S., Hohng, Sungchul, Hong, Seok Cheol, Hugel, Thorsten, Ingargiola, Antonino, Joo, Chirlmin, Kapanidis, Achillefs N., Kim, Harold D., Laurence, Ted, Lee, Nam Ki, Lee, Tae Hee, Lemke, Edward A., Margeat, Emmanuel, Michaelis, Jens, Michalet, Xavier, Myong, Sua, Nettels, Daniel, Peulen, Thomas Otavio, Ploetz, Evelyn, Razvag, Yair, Robb, Nicole C., Schuler, Benjamin, Soleimaninejad, Hamid, Tang, Chun, Vafabakhsh, Reza, Lamb, Don C., Seidel, Claus A.M., Weiss, Shimon, Boudker, Olga, Lerner, Eitan, Barth, Anders, Hendrix, Jelle, Ambrose, Benjamin, Birkedal, Victoria, Blanchard, Scott C., Börner, Richard, Chung, Hoi Sung, Cordes, Thorben, Craggs, Timothy D., Deniz, Ashok A., Diao, Jiajia, Fei, Jingyi, Gonzalez, Ruben L., Gopich, Irina V., Ha, Taekjip, Hanke, Christian A., Haran, Gilad, Hatzakis, Nikos S., Hohng, Sungchul, Hong, Seok Cheol, Hugel, Thorsten, Ingargiola, Antonino, Joo, Chirlmin, Kapanidis, Achillefs N., Kim, Harold D., Laurence, Ted, Lee, Nam Ki, Lee, Tae Hee, Lemke, Edward A., Margeat, Emmanuel, Michaelis, Jens, Michalet, Xavier, Myong, Sua, Nettels, Daniel, Peulen, Thomas Otavio, Ploetz, Evelyn, Razvag, Yair, Robb, Nicole C., Schuler, Benjamin, Soleimaninejad, Hamid, Tang, Chun, Vafabakhsh, Reza, Lamb, Don C., Seidel, Claus A.M., Weiss, Shimon, and Boudker, Olga

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.

Published
2021

31. Interplay of [alpha]-synuclein binding and conformational switching probed by single-molecule fluorescence

Author

Ferreon, Allan Chris M., Gambin, Yann, Lemke, Edward A., and Deniz, Ashok A.

Subjects
Binding proteins -- Health aspects, Binding proteins -- Physiological aspects, Fluorescence -- Usage, Science and technology
Abstract

We studied the coupled binding and folding of [alpha]-synuclein, an intrinsically disordered protein linked with Parkinson's disease. Using single-molecule fluorescence resonance energy transfer and correlation methods, we directly probed protein membrane association, structural distributions, and dynamics. Results revealed an intricate energy landscape on which binding of [alpha]-synuclein to amphiphilic small molecules or membrane-like partners modulates conformational transitions between a natively unfolded state and multiple [alpha]-helical structures. [alpha]-Synuclein conformation is not continuously tunable, but instead partitions into 2 main classes of folding landscape structural minima. The switch between a broken and an extended helical structure can be triggered by changing the concentration of binding partners or by varying the curvature of the binding surfaces presented by micelles or bilayers composed of the lipid-mimetic SDS. Single-molecule experiments with lipid vesicles of various composition showed that a low fraction of negatively charged lipids, similar to that found in biological membranes, was sufficient to drive [alpha]-synuclein binding and folding, resulting here in the induction of an extended helical structure. Overall, our results imply that the 2 folded structures are preen-coded by the [alpha]-synuclein amino acid sequence, and are tunable by small-molecule supramolecular states and differing membrane properties, suggesting novel control elements for biological and amyloid regulation of [alpha]-synuclein. amyloid | misfolding | Parkinson's disease | protein folding | supramolecular

Published
2009

34. FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices

Author

Lerner, Eitan, primary, Barth, Anders, additional, Hendrix, Jelle, additional, Ambrose, Benjamin, additional, Birkedal, Victoria, additional, Blanchard, Scott C, additional, Börner, Richard, additional, Sung Chung, Hoi, additional, Cordes, Thorben, additional, Craggs, Timothy D, additional, Deniz, Ashok A, additional, Diao, Jiajie, additional, Fei, Jingyi, additional, Gonzalez, Ruben L, additional, Gopich, Irina V, additional, Ha, Taekjip, additional, Hanke, Christian A, additional, Haran, Gilad, additional, Hatzakis, Nikos S, additional, Hohng, Sungchul, additional, Hong, Seok-Cheol, additional, Hugel, Thorsten, additional, Ingargiola, Antonino, additional, Joo, Chirlmin, additional, Kapanidis, Achillefs N, additional, Kim, Harold D, additional, Laurence, Ted, additional, Lee, Nam Ki, additional, Lee, Tae-Hee, additional, Lemke, Edward A, additional, Margeat, Emmanuel, additional, Michaelis, Jens, additional, Michalet, Xavier, additional, Myong, Sua, additional, Nettels, Daniel, additional, Peulen, Thomas-Otavio, additional, Ploetz, Evelyn, additional, Razvag, Yair, additional, Robb, Nicole C, additional, Schuler, Benjamin, additional, Soleimaninejad, Hamid, additional, Tang, Chun, additional, Vafabakhsh, Reza, additional, Lamb, Don C, additional, Seidel, Claus AM, additional, and Weiss, Shimon, additional

Published
2021
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35. FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices

Author

Lerner, Eitan, Barth, Anders, Hendrix, Jelle, Ambrose, Benjamin, Birkedal, Victoria, Blanchard, Scott C., Börner, Richard, Chung, Hoi Sung, Cordes, Thorben, Craggs, Timothy D., Deniz, Ashok A., Diao, Jiajia, Fei, Jingyi, Gonzalez, Ruben L., Gopich, Irina V., Ha, Taekjip, Hanke, Christian A., Haran, Gilad, Hatzakis, Nikos S., Hohng, Sungchul, Hong, Seok Cheol, Hugel, Thorsten, Ingargiola, Antonino, Joo, Chirlmin, Kapanidis, Achillefs N., Kim, Harold D., Laurence, Ted, Lee, Nam Ki, Lee, Tae Hee, Lemke, Edward A., Margeat, Emmanuel, Michaelis, Jens, Michalet, Xavier, Myong, Sua, Nettels, Daniel, Peulen, Thomas Otavio, Ploetz, Evelyn, Razvag, Yair, Robb, Nicole C., Schuler, Benjamin, Soleimaninejad, Hamid, Tang, Chun, Vafabakhsh, Reza, Lamb, Don C., Seidel, Claus A.M., Weiss, Shimon, Boudker, Olga, Institute of Chemistry, The Hebrew University of Jerusalem, The Hebrew University of Jerusalem (HUJ), Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], Hasselt University (UHasselt), University of Sheffield [Sheffield], Aarhus University [Aarhus], St Jude Children's Research Hospital, Hochschule Mittweida - University of Applied Sciences, National Institutes of Health [Bethesda] (NIH), Ludwig-Maximilians-Universität München (LMU), The Scripps Research Institute [La Jolla], University of California [San Diego] (UC San Diego), University of California-University of California, University of Cincinnati (UC), University of Chicago, Columbia University [New York], Howard Hughes Medical Institute (HHMI), Weizmann Institute of Science [Rehovot, Israël], University of Copenhagen = Københavns Universitet (KU), Seoul National University [Seoul] (SNU), Korea University [Seoul], University of Freiburg [Freiburg], University of California [Los Angeles] (UCLA), University of California, Delft University of Technology (TU Delft), University of Oxford [Oxford], Georgia Institute of Technology [Atlanta], Lawrence Livermore National Laboratory (LLNL), Pennsylvania State University (Penn State), Penn State System, University Medical Center of the Johannes Gutenberg-University Mainz, Institute of Molecular Biology (IMB), Johannes Gutenberg - Universität Mainz (JGU), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Ulm (UUlm), Johns Hopkins University (JHU), Université de Zurich [Switzerland], University of California [San Francisco] (UCSF), University of Warwick [Coventry], University of Melbourne, Peking University [Beijing], Northwestern University [Evanston], The Scripps Research Institute [La Jolla, San Diego], University of Copenhagen = Københavns Universitet (UCPH), University of California (UC), University of Oxford, Johannes Gutenberg - Universität Mainz = Johannes Gutenberg University (JGU), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), University of California [San Francisco] (UC San Francisco), and Margeat, Emmanuel

Subjects
0301 basic medicine, conformation, Open science, Computer science, Structural Biology and Molecular Biophysics, AMINOACYL-TRANSFER-RNA, INTRAMOLECULAR DISTANCE DISTRIBUTIONS, Review Article, RESONANCE ENERGY-TRANSFER, 01 natural sciences, biomolecules, FREELY DIFFUSING MOLECULES, Documentation, Fluorescence Resonance Energy Transfer, Mainstream, structural biology, Biology (General), General Neuroscience, NANO-POSITIONING SYSTEM, General Medicine, dynamics, INTRINSICALLY DISORDERED PROTEINS, Single Molecule Imaging, FLUORESCENCE CORRELATION SPECTROSCOPY, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Medicine, community, single-molecule, QH301-705.5, Science, Appeal, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Bioengineering, chemical biology, 010402 general chemistry, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, ALTERNATING-LASER EXCITATION, Biochemistry and Chemical Biology, molecular biophysics, biochemistry, Molecular Biology, Structure (mathematical logic), General Immunology and Microbiology, SINGLE-MOLECULE FRET, TRANSITION PATH TIMES, Data science, 0104 chemical sciences, 030104 developmental biology, FRET, Position paper, Generic health relevance, Biochemistry and Cell Biology
Abstract

International audience; 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.

Published
2020

39. PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins

Author

Lazar, Tamas, primary, Martínez-Pérez, Elizabeth, additional, Quaglia, Federica, additional, Hatos, András, additional, Chemes, Lucía B, additional, Iserte, Javier A, additional, Méndez, Nicolás A, additional, Garrone, Nicolás A, additional, Saldaño, Tadeo E, additional, Marchetti, Julia, additional, Rueda, Ana Julia Velez, additional, Bernadó, Pau, additional, Blackledge, Martin, additional, Cordeiro, Tiago N, additional, Fagerberg, Eric, additional, Forman-Kay, Julie D, additional, Fornasari, Maria S, additional, Gibson, Toby J, additional, Gomes, Gregory-Neal W, additional, Gradinaru, Claudiu C, additional, Head-Gordon, Teresa, additional, Jensen, Malene Ringkjøbing, additional, Lemke, Edward A, additional, Longhi, Sonia, additional, Marino-Buslje, Cristina, additional, Minervini, Giovanni, additional, Mittag, Tanja, additional, Monzon, Alexander Miguel, additional, Pappu, Rohit V, additional, Parisi, Gustavo, additional, Ricard-Blum, Sylvie, additional, Ruff, Kiersten M, additional, Salladini, Edoardo, additional, Skepö, Marie, additional, Svergun, Dmitri, additional, Vallet, Sylvain D, additional, Varadi, Mihaly, additional, Tompa, Peter, additional, Tosatto, Silvio C E, additional, and Piovesan, Damiano, additional

Published
2020
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48. Molecular determinants of large cargo transport into the nucleus

Author

Paci, Giulia and Lemke, Edward A

Subjects
medicine.anatomical_structure, Chemistry, Cytoplasm, Kinetics, medicine, Biophysics, NLS, Nuclear pore, Multivalent binding, Nuclear transport, Receptor, Nucleus
Abstract

Transport of molecules between the nucleus and the cytoplasm is tightly regulated by the nuclear pore complex (NPC). Even very large cargoes such as many pathogens, mRNAs and pre-ribosomal subunits can pass the NPC intact. Compared to small import complexes, for such large cargoes >15 nm there is very little quantitative understanding of the mechanism for efficient transport, the role of multivalent binding to nuclear transport receptors via nuclear localisation sequences (NLSs) and effects of size differences. Here, we assayed nuclear import kinetics in cells for a total of 30 large cargo models based on four capsid-like particles in the size range of 17-36 nm, with tuneable numbers of up to 240 NLSs. We show that the requirements for transport scale non-linearly with size and obey a minimal cut off of functional import requiring more than 10 NLS in the lowest case. Together, our results reveal the key molecular determinants on large cargo import kinetics in cells.

Published
2019

50. Synthesis of Azido-Glycans for Chemical Glycomodification of Proteins

Author

Wawryszyn, Mirella, Sauter, Paul F., Nieger, Martin, Koos, Martin R. M., Koehler, Christine, Luy, Burkhard, Lemke, Edward A., Bräse, Stefan, and Department of Chemistry

Subjects
DESIGNER PROTEINS, COUPLING-CONSTANTS, High-Mannose Motif, GLYCOSYLATION, 116 Chemical sciences, Oligosaccharides, EFFICIENT, FC-GAMMA-RIII, carbohydrates (lipids), N-Glycans, HIGH-MANNOSE-TYPE, Synthetic methods, Glycoforms, CORE FUCOSE, CHEMOENZYMATIC SYNTHESIS, GENETIC-CODE EXPANSION, Glycoproteins
Abstract

Chemically produced, accurately linkable oligosaccharides are of importance for the synthesis of neo-glycoproteins. On the route to high-mannose type N-glycans, we present a convenient synthesis of several glycans bearing an azide moiety at the reducing end. An azido-glycan core structure as valuable precursor was modified into the protected N-glycan pentasaccharide core structure and the possibility of modular attachment of different antenna was demonstrated through synthesis of a pentamannose donor and glycosylation with the core structure. The azido function allows for chemical ligation with recombinantly modified proteins featuring noncanonical cyclooctyne amino acids, providing access to customized glycopatterns of glycoproteins, e.g., of antibodies that are of high interest for biopharmaceutical applications.

Published
2018

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