Search

Your search keyword '"Lemke, Edward A"' showing total 7 results
Start Over Author "Lemke, Edward A" Language undetermined
7 results on '"Lemke, Edward A"'

1. 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, Cordes, Thorben, Agam, Ganesh [0000-0003-1623-4920], Gebhardt, Christian [0000-0002-5834-6579], Popara, Milana [0000-0003-2626-6096], Folz, Julian [0000-0001-8232-8787], Ambrose, Benjamin [0000-0002-5046-2532], Chamachi, Neharika [0000-0002-4657-9092], Craggs, Timothy D [0000-0002-7121-0609], Grohmann, Dina [0000-0002-0570-2517], Ha, Taekjip [0000-0003-2195-6258], Hartmann, Andreas [0000-0002-2213-2763], Hendrix, Jelle [0000-0001-5731-1297], Hugel, Thorsten [0000-0003-3292-4569], Kang, Hyun-Seo [0000-0003-4029-5096], Krainer, Georg [0000-0002-9626-7636], Lemke, Edward A [0000-0002-0634-0503], Lerner, Eitan [0000-0002-3791-5277], Margeat, Emmanuel [0000-0001-6063-6420], Michaelis, Jens [0000-0002-2739-4172], Moya Muñoz, Gabriel G [0000-0003-2797-2005], Quast, Robert B [0000-0001-8166-6952], Schlierf, Michael [0000-0002-6209-2364], Schröder, Tim [0000-0002-2474-5395], Thurn, Johann [0000-0001-6370-7052], van Noort, John [0000-0002-9094-5598], Weiss, Shimon [0000-0002-0720-5426], Wendler, Nicolas [0000-0002-5436-3929], Barth, Anders [0000-0003-3671-3072], Seidel, Claus AM [0000-0002-5171-149X], Lamb, Don C [0000-0002-0232-1903], Cordes, Thorben [0000-0002-8598-5499], Apollo - University of Cambridge Repository, Agam, Ganesh/0000-0003-1623-4920, Popara, Milana/0000-0003-2626-6096, Chamachi, Neharika/0000-0002-4657-9092, Quast, Robert, Benjamin/0000-0001-8166-6952, 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., and Cordes, Thorben

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

Funder: Ludwig Maximilians University Munich | Center for NanoScience, Ludwig-Maximilians-Universität Mnchen (CeNS, LMU); doi: https://doi.org/10.13039/501100007153, Funder: Alexander von Humboldt-Stiftung (Alexander von Humboldt Foundation); doi: https://doi.org/10.13039/100005156, 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
Full Text
View/download PDF

2. 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

3. 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

4. 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
Technology, ComputingMilieux_LEGALASPECTSOFCOMPUTING, Biological Sciences, Medical and Health Sciences, Developmental Biology
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

5. Architecture of TAF11/TAF13/TBP complex suggests novel regulation properties of general transcription factor TFIID

Author

Gupta, Kapil, Watson, Aleksandra A, Baptista, Tiago, Scheer, Elisabeth, Chambers, Anna L, Koehler, Christine, Zou, Juan, Obong-Ebong, Ima, Kandiah, Eaazhisai, Temblador, Arturo, Round, Adam, Forest, Eric, Man, Petr, Bieniossek, Christoph, Laue, Ernest D, Lemke, Edward A, Rappsilber, Juri, Robinson, Carol V, Devys, Didier, Tora, Làszlò, Berger, Imre, European Molecular Biology Laboratory [Grenoble] (EMBL), Department of Biochemistry [Cambridge], University of Cambridge [UK] (CAM), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), BrisSynBio Centre, The School of Biochemistry, University of Bristol [Bristol], European Molecular Biology Laboratory [Heidelberg] (EMBL), Wellcome Trust Centre for Cell Biology, University of Edinburgh, Physical and Theoretical ChemistryLaboratory, University of Oxford [Oxford], Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institute of Microbiology of the Czech Academy of Sciences (MBU / CAS), Czech Academy of Sciences [Prague] (CAS), Department of Chemistry, University of Oxford, University of Oxford [Oxford]-Chemistry Research Laboratory, University of Oxford, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institute of Microbiology of the Czech Academy of Sciences [Prague, Czech Republic] (MBU / CAS), University of Oxford-Chemistry Research Laboratory, Chambers, Anna L [0000-0001-8133-6240], Temblador, Arturo [0000-0003-3076-6317], Laue, Ernest D [0000-0002-7476-4148], Devys, Didier [0000-0001-9655-3512], Tora, Làszlò [0000-0001-7398-2250], Berger, Imre [0000-0001-7518-9045], and Apollo - University of Cambridge Repository

Subjects
MESH: TFIID, Protein Conformation, Structural Biology and Molecular Biophysics, genetic processes, MESH: biophysics, S. cerevisiae, MESH: biochemistry, MESH: transcription factors, Crystallography, X-Ray, TBP associated factors, Mass Spectrometry, MESH: histone fold domain, MESH: human, biophysics, Protein Interaction Mapping, structural biology, Biology (General), Promoter Regions, Genetic, Histone Acetyltransferases, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], TFIID, MESH: TBP associated factors, Medicine, Protein Binding, Research Article, Human, QH301-705.5, MESH: structural biology, Science, information science, MESH: core promoter DNA, macromolecular substances, histone fold domain, Biochemistry and Chemical Biology, transcription factors, biochemistry, Humans, MESH: gene regulation, TATA-Binding Protein Associated Factors, Bristol BioDesign Institute, core promoter DNA, DNA, TATA-Box Binding Protein, SYNTHETIC BIOLOGY, enzymes and coenzymes (carbohydrates), health occupations, MESH: S. cerevisiae, Transcription Factor TFIID, gene regulation
Abstract

International audience; General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-interaction domain (CTID) in TAF13, which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.

Published
2017

6. The CD27L and CTP1L Endolysins Targeting Clostridia Contain a Built-in Trigger and Release Factor

Author

Zhang, Gongyi, Mertens, Haydyn, Garefalaki, Vasiliki, Jeffries, Cy, Thompson, Andrew, Lemke, Edward A., Svergun, Dmitri, Mayer, Melinda J., Narbad, Arjan, and Meijers, Rob

Subjects
Lysis, Lysin, Pathogenesis, Crystallography, X-Ray, Pathology and Laboratory Medicine, Biochemistry, Bacteriophage, Protein structure, Medicine and Health Sciences, Bacteriophages, Biology (General), Enzyme Chemistry, Enzyme Precursors, 0303 health sciences, biology, Enzymes, Bacterial Pathogens, 3. Good health, Medical Microbiology, Viral Enzymes, Host-Pathogen Interactions, Release factor, Research Article, Protein Structure, QH301-705.5, Immunology, Models, Biological, Microbiology, Enzyme Regulation, Viral Proteins, 03 medical and health sciences, Microbial Control, Virology, Endopeptidases, Hydrolase, Genetics, Humans, ddc:610, Microbial Pathogens, Molecular Biology, 030304 developmental biology, Enzyme Kinetics, Clostridioides difficile, 030306 microbiology, Biology and Life Sciences, Proteins, RC581-607, biology.organism_classification, Protein Structure, Tertiary, Regulatory Proteins, Enzyme Structure, Enzymology, Biocatalysis, Biophysics, Molecular Complexes, Parasitology, Immunologic diseases. Allergy, Linker, Alpha helix
Abstract

The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall., Author Summary Clostridium difficile infection is a common cause of hospital-acquired diarrhea, following broad-spectrum antibiotic treatment particularly in elderly patients. Bacteriophage therapy could provide an alternative treatment, but a better understanding of the viral components that lyse the bacterial cell is necessary. Here, we report on the activation of two endolysins from bacteriophages that lyse Clostridia. The structures of autoproteolytic fragments of two endolysins were determined by X-ray crystallography. Based on the structures, we introduced mutations that affect the autolytic cleavage of the enzymatic portion of the endolysins, and we show that two oligomeric states have an effect on the cleavage mechanism. Moreover, the lysis activity is affected when autocleavage is inhibited for one endolysin. We propose that the cleavage and oligomerization are linked, and they provide the endolysin with a trigger and release mechanism that leads to activation. The identification of a trigger and release factor may not only be relevant to Clostridia endolysins, but could be an important factor in the triggering of many bacteriophage endolysins. A fuller understanding of this activation mechanism will help in the design of recombinant endolysins or bacteriophages with a more efficient therapeutic potential.

Published
2014

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

Author

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

Subjects
bepress|Physical Sciences and Mathematics|Physics, Biological Physics (physics.bio-ph), bepress|Life Sciences|Biology, FOS: Biological sciences, biological sciences, FOS: Physical sciences, Physics - Biological Physics, Quantitative Biology - Quantitative Methods, Quantitative Methods (q-bio.QM)
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

Catalog

Books, media, physical & digital resources
See catalog results