In recent years, single-molecule Förster resonance energy transfer (smFRET) has emerged as a critical and flexible tool in structural biology, particularly in the study of highly dynamic regions and molecular assemblies. The usefulness of smFRET can be further extended by combining it with computational approaches, marrying the coarse-grained experimental data with higher-resolution in silico calculations. Here we use smFRET to determine six pairwise distances within the intrinsically disordered C-terminal domain of the troponin I subunit (TnIC) of the cardiac troponin complex. We used published conflicting structures of TnIC as starting models for molecular dynamics simulations, which were validated through successful comparison with smFRET measurements before extracting information on conformational dynamics. We flnd that pairwise distances between residues fluctuate widely in silico, but simulations are generally in good agreement with longer time scale smFRET measurements after averaging across time. Finally, Monte Carlo simulations establish that the lower-energy conformers of TnIC are indeed varied, but that the highest-sampled clusters resemble the published, conflicting models. In this way, we flnd that the controversial structures are simply stabilized local minima of this dynamic region, and a population including all three would still be consistent with spectroscopic measurements. Taken together, the combined approaches described here allow us to critically evaluate existing models of TnIC, giving insight into the conformation and dynamics of TnIC’s disordered state prior to its probable disorder–order transition. Moreover, they provide a framework for combining computational and experimental methods with diflerent time scales for the study of disordered and dynamic protein states.
We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ET) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ET between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament.
Using a new Titan Krios stage equipped with a single-axis holder, we developed two methods to accelerate the collection of tilt-series. We demonstrate a continuous-tilting method that can record a tilt-series in seconds, but with loss of details finer than ~4 nm. We also demonstrate a fastincremental method that can record a tilt-series several-fold faster than current methods and with similar resolution. We characterize the utility of both methods in real biological electron cryotomography workflows. We identify opportunities for further improvements in hardware and software and speculate on the impact such advances could have on structural biology. Highlights Continuous-tilting: acquire tilt-series in seconds Currently limited to an estimated 4 nm resolution Fast-incremental: acquire tilt-series several-fold faster than current methods Can produce high quality subtomogram average at a fraction of the time
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