Quantitative Connection Between Ensemble Thermodynamics and Single-Molecule Kinetics: A Case Study Using Cryogenic Electron Microscopy and Single-Molecule Fluorescence Resonance Energy Transfer Investigations of the Ribosome

摘要

At equilibrium, thermodynamic and kinetic information can be extracted frombiomolecular energy landscapes by many techniques. However, while static,ensemble techniques yield thermodynamic data, often only dynamic,single-molecule techniques can yield the kinetic data that describestransition-state energy barriers. Here we present a generalized framework basedupon dwell-time distributions that can be used to connect such static, ensembletechniques with dynamic, single-molecule techniques, and thus characterizeenergy landscapes to greater resolutions. We demonstrate the utility of thisframework by applying it to cryogenic electron microscopy (cryo-EM) andsingle-molecule fluorescence resonance energy transfer (smFRET) studies of thebacterial ribosomal pre-translocation complex. Among other benefits,application of this framework to these data explains why two transient,intermediate conformations of the pre-translocation complex, which are observedin a cryo-EM study, may not be observed in several smFRET studies.