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Long-range (>10 μm) transport of electrons along networks of Geobacter sulfurreducens protein filaments, known as microbial nanowires, has been invoked to explain a wide range of globally important redox phenomena. These nanowires were previously thought to be type IV pili composed of PilA protein. Here, we report a 3.7 Å resolution cryoelectron microscopy structure, which surprisingly reveals that, rather than PilA, G. sulfurreducens nanowires are assembled by micrometer-long polymerization of the hexaheme cytochrome OmcS, with hemes packed within ∼3.5-6 Å of each other. The inter-subunit interfaces show unique structural elements such as inter-subunit parallel-stacked hemes and axial coordination of heme by histidines from neighboring subunits. Wild-type OmcS filaments show 100-fold greater conductivity than other filaments from a ΔomcS strain, highlighting the importance of OmcS to conductivity in these nanowires. This structure explains the remarkable capacity of soil bacteria to transport electrons to remote electron acceptors for respiration and energy sharing.

Cryo‐electron microscopy (cryo‐EM) has transformed structural biology over the past 12 years, with it now being routine rather than exceptional to reach a near‐atomic level of resolution for proteins and macromolecular complexes. Samples are immobilized by vitrification and this sample can be maintained at liquid nitrogen temperatures in the vacuum of the electron microscope with negligible sublimation. Due to the low electron doses needed to avoid radiation damage, averaging over tens of thousands to hundreds of thousands of particle images is used to achieve a high signal‐to‐noise ratio. An alternative approach has been proposed where samples are at room temperature in the liquid state, maintained in the vacuum of the electron microscope by thin film enclosures that are relatively transparent to electrons while preventing evaporation of the liquid. A paper has argued that using this liquid‐phase approach, higher resolution (3.2 Å) can be achieved than using cryo‐EM (3.4 Å) when imaging and reconstructing adeno‐associated virus particles. I show here that these assertions are untrue, and that basic principles in mathematics and physics would need to be violated to achieve the stated resolution in the liquid state. Thus, high resolution liquid phase EM of macromolecules remains science fiction. [ABSTRACT FROM AUTHOR]

Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, "open," and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex.

A stepwise addition protocol was developed to display cargo using bacteriophage P22 capsids and the phage decoration (Dec) protein. Three-dimensional image reconstructions of frozen-hydrated samples of P22 particles with nanogold-labeled Dec bound to them revealed the locations of the N- and C-termini of Dec. Each terminus is readily accessible for molecular display through affinity tags such as nickel-nitrilotriacetic acid, providing a total of 240 cargo-binding sites. Dec was shown by circular dichroism to be a β-sheet rich protein, and fluorescence anisotropy binding experiments demonstrated that Dec binds to P22 heads with high (~110 nm) affinity. Dec also binds to P22 nanotubes, which are helically symmetric assemblies that form when the P22 coat protein contains the F170A amino acid substitution. Several classes of tubes with Dec bound to them were visualized by cryo-electron microscopy and their three-dimensional structures were determined by helical reconstruction methods. In all instances, Dec trimers bound to P22 capsids and nanotubes at positions where three neighboring capsomers (oligomers of six coat protein subunits) lie in close proximity to one another. Stable interactions between Dec and P22 allow for the development of robust, nanoscale size, display vehicles.

Previous studies showed that the K342E substitution in the Saccharomyces cerevisiae Rad51 protein increases the interaction with Rad54 protein in the two-hybrid system, leads to increased sensitivity to the alkylating agent MMS and hyper-recombination in an oligonucleotide-mediated gene targeting assay. K342 localizes in loop 2, a region of Rad51 whose function is not well understood. Here, we show that Rad51-K342E displays DNA-independent and DNA-dependent ATPase activities, owing to its ability to form filaments in the absence of a DNA lattice. These filaments exhibit a compressed pitch of 81 A, whereas filaments of wild-type Rad51 and Rad51-K342E on DNA form extended filaments with a 97 A pitch. Rad51-K342E shows near normal binding to ssDNA, but displays a defect in dsDNA binding, resulting in less stable protein-dsDNA complexes. The mutant protein is capable of catalyzing the DNA strand exchange reaction and is insensitive to inhibition by the early addition of dsDNA. Wild-type Rad51 protein is inhibited under such conditions, because of its ability to bind dsDNA. No significant changes in the interaction between Rad51-K342E and Rad54 could be identified. These findings suggest that loop 2 contributes to the primary DNA-binding site in Rad51, controlling filament formation and ATPase activity.