Type 1 pili are filamentous protein assemblies on the surface of Gram‐negative bacteria that mediate adhesion to host cells during the infection process. The molecular structure of type 1 pili remains elusive on the atomic scale owing to their insolubility and noncrystallinity. Herein we describe an approach for hybrid‐structure determination that is based on data from solution‐state NMR spectroscopy on the soluble subunit and solid‐state NMR spectroscopy and STEM data on the assembled pilus. Our approach is based on iterative modeling driven by structural information extracted from different sources and provides a general tool to access pseudo atomic structures of protein assemblies with complex subunit folds. By using this methodology, we determined the local conformation of the FimA pilus subunit in the context of the assembled type 1 pilus, determined the exact helical pilus architecture, and elucidated the intermolecular interfaces contributing to pilus assembly and stability with atomic detail.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.