Electron Microscopy Identifies Protein Conformational Substates in Liquid Environment

Abstract Protein functions are closely linked to their dynamic behaviors, particularly involving low-abundance transient states. Quantifying these interconversions remains experimentally challenging, especially for proteins containing intrinsically disordered regions (IDRs). In this study, we utilize low-dose electron microscopy using graphene liquid cells to image and quantify interconversions between protein conformational states under optimized electron beam conditions and confinement effects. Applied to four proteins in solution: CRISPR-associated protein (Cas9), DNA binding protein (GapR), restriction enzyme (EcoRI), and an RNA binding protein with IDRs (FUS), we visualized low-abundance transient structures distinct from crystalline forms and elucidated transition pathways which are validated by cryo-electron microscopy, revealing differences in their energy landscapes through single-molecule imaging. We analyzed domain-scale rearrangements at ∼1–2 nm effective conformational resolution, cross-validated against independent computational and experimental methods. Shifts in state equilibria and conformational adaptations were observed when proteins are mutated or during biomolecular interactions, such as target searching, binding, and cleavage with nucleic acids. Notably, changes in the energy landscape were observed when proteins with IDRs and RNA were within close proximity but not in apparent contact, suggesting that conformational redistribution may precede complete binding. This study shows how to characterize protein conformations and their time-dependent changes using instrumentation that is widely available at research universities but not yet used for these purposes.