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Democratizing single-molecule FRET for Dynamic Structural Biology: New tools and an integrative approach to understanding DNA dynamics - 1 February 2022

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Speaker Dr Tim Craggs, Department of Chemistry, University of Sheffield

Host Julien Bergeron

Abstract Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between dynamic biomolecules. In the first part of this talk, I will present the smfBox (1) and its evolution the EI-FLEX (2), both robust and economic confocal smFRET microscopes, and show how you can use them for accurate absolute FRET measurements (reproducing the FRET efficiencies from our multi-lab benchmarking study (3) and measuring biomolecular conformational dynamics (exemplified by DNA hairpin opening and closing).

I will then give an example of our iterative approach to determining conformational ensembles of biomolecules, in which we combined our smFRET experiments with molecular dynamic simulations to reveal the structure and dynamics of the DNA-protein complex formed between gapped-DNA and DNA Polymerase I (4). This led us to propose a general mechanism for substrate recognition by structure-specific DNA enzymes driven by protein sensing of the conformational dynamics of their substrates.

Finally, I will present a selection of unpublished results from the lab including: (i) Quantitative Quenchable FRET (qqFRET), a new single-molecule method sensitive to structural changes below 3 nm, (ii) iMotif DNA dynamics and (iii) new work on DNA minicircles.


  1. Ambrose, B., Baxter, J.M., Cully, J. et al. The smfBox is an open-source platform for single-molecule FRET. Nat Commun 11, 5641 (2020).




  1. Hellenkamp, B. et al. Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study. Nature Methods 15, 669-676 (2018).


  1. Craggs, T. D. et al. Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase. Nucleic Acids Res 47, 10788-10800 (2019).

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