Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

16-POS

Board 8

COMBINED CRYO-EM AND SINGLE-MOLECULE FRET APPROACH FOR STUDYING CONFORMATIONAL SPECTRA OF MSBA Sylwia Dylnicka 1 ; Anika Baier 1 ; Rainer Kurre 2,3 ; Changjiang You 2,3 ; Jacob Piehler 2,3 ; Dovile Januliene 1,2 ; Arne Moeller 1,2 ; 1 Osnabrück University, Department of Biology/Chemistry, Structural Biology Section, Osnabrück, Germany 2 Center of Cellular Nanoanalytics Osnabrück (CellNanOs), Osnabrück , Germany 3 Osnabrück University, Department of Biology/Chemistry, Biophysics Section, Osnabrück, Germany MsbA is an ATP-binding cassette (ABC) transporter that undergoes large conformational rearrangements during its transport cycle. Cryo-EM studies captured MsbA in multiple conformations across a variety of membrane-mimetics, including detergents and different nanodisc assemblies, highlighting the profound effect of the experimental environment can have on the observed conformational ensembles. In this work, we combined cryo-EM and single molecule Förster resonance energy transfer (smFRET) to investigate the conformational spectra of nucleotide-bound MsbA in different detergents. While both techniques allow to study the distribution of conformational states, smFRET can additionally provide a dimension of time. However, before adding the fourth dimension, we first need to benchmark smFRET against our cryo-EM structures. For this, we introduced a donor–acceptor fluorophore pair at residue D277 of the homo-dimer E. coli MsbA, allowing us to monitor the opening and closure of the extracellular gate and compared the particle distribution between the two outward-facing conformations (OF open and OF closed ) upon trapping with ATP vanadate in two different detergents. Both cryo-EM and smFRET analysis consistently showed that in DDM MsbA predominantly adopts an OF open conformation, while in LMNG, it exists in a mixed population of OF open and OF closed conformations in a similar ratio. Together, this study emphasizes the importance of the membrane-mimetic environment on the conformational spectrum of nucleotide-bound MsbA and lays the foundation for combining complementary cryo-EM and smFRET approaches for interpreting MsbA behavior.

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