Biology subjects

Sofos, N.

Publications and source records attributed to Sofos, N..

7 recordsLinked to original sources

bioRxiv2026

Genome delivery of a contractile tailed phage and its superinfection exclusion mechanism

Successful viral infection requires efficient adsorption to the target cell, followed by membrane penetration for genome translocation into the host cytoplasm. Bacteriophage T4 initiates infection by binding to Escherichia coli receptors, triggering sequential conformational changes that culminate in genome delivery through viral channels spanning the host cell envelope. Here, we resolved structures of bacteriophage T4 at discrete stages. This revealed how long tail fiber extension affects the baseplate to initiate tail contraction, and the channel formation by the tape measure protein for genome translocation. We further demonstrate that the virus-encoded superinfection exclusion protein Imm binds to the tape measure protein to prevent secondary infections. Our findings offer insights into the coordinated process of genome delivery and phage-encoded superinfection exclusion proteins that prevent genome translocation.

microbiology↗

bioRxiv2025

Structural characterization of an extracellular contractile injection system from Photorhabdus luminescens in extended and contracted states.

Contractile injection systems (CISs) are phage-tail-like nanosyringes that mediate bacterial interactions by puncturing target cell membranes. Within these systems, Photorhabdus Virulence Cassettes (PVCs) can translocate toxins across eukaryotic target cell membranes. The structure of a PVC has been described at atomic level and engineered to deliver diverse protein cargoes into non-natively-targeted organisms. Despite the structural insights into several CISs, information on PVCs from other species and details on the contraction mechanism remain limited. Here, we present the single-particle cryo-electron microscopy structure of PlPVC1, a PVC from the nematode symbiont and insect pathogen Photorhabdus luminescens DJC, in both extended and contracted states. Our structure displays distinct structural features that differ from other CISs, such as a cage surrounding the central spike, a larger sheath adaptor, and a plug exposed to the tube lumen. Moreover, we present the structures of the PlPVC1 fiber as well as the baseplate of the contracted particle, yielding insight into the contraction mechanism. This study provides structural details of the contracted state of the PlPVC1 particle and supports the model in which contraction is triggered. Furthermore, it facilitates the comparison of PlPVC1 with other contractile systems and expands the scope of engineering opportunities for future biomedical and biotechnological applications.

molecular biology↗

bioRxiv2024

Towards a complete phage tail fiber structure atlas.

Bacteriophages use receptor-binding proteins (RBPs) to adhere to bacterial hosts. Understanding the structure of these RBPs can provide insights into their target interactions. Tail fibers, a prominent type of RBP, are typically elongated, flexible, and trimeric proteins, making it challenging to obtain high-resolution experimental data of their full-length structures. Recent advancements in deep learning-based protein structure prediction, such as AlphaFold2-multimer (AF2M) and ESMfold, allow for the generation of high-confidence predicted models of complete tail fibers. In this paper, we introduce RBPseg, a method that combines monomeric ESMfold predictions with a novel sigmoid distance pair (sDp) protein segmentation technique. This method segments the tail fiber sequences into smaller fractions, preserving domain boundaries. These segments are then predicted in parallel using AF2M and assembled into a full fiber model. We demonstrate that RBPseg significantly improves AF2M v2.3.2 in terms of model confidence, running time, and memory usage. To validate our approach, we used single-particle cryo-electron microscopy to analyze five tail fibers from three phages of the BASEL collection. Additionally, we conducted a structural classification of 67 fibers and their domains, which identified 16 well-defined tail fiber classes and 89 domains. Our findings suggest the existence of modular fibers as well as fibers with different sequences and shared structure, indicating possible sequence convergence, divergence, and domain swapping. We further demonstrate that these structural classes account for at least 24% of the known tail fiber universe.

microbiology↗

bioRxiv2022

Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon

CRISPR-associated transposons (CASTs) represent unique mobile genetic elements that co-opted CRISPR-Cas immune systems for RNA-guided DNA transposition. Type V-K CAST is composed by Cas12k, TniQ, TnsC and TnsB. Here, we present the 2.46 [A] cryoelectron microscopy structure of the Scytonema hofmannii CAST TnsB transposase in complex with the strand transfer DNA in a post-catalytic state. The shTnsB strand transfer complex maintains the intertwined architecture of the MuA phage transpososome. However, the building of the assembly depends on different local interactions. The protein-DNA complex forms a pseudo-symmetrical assembly in which the 4 protomers of shTnsB adopt two different conformations. The recognition of the transposon ends is accomplished by two small helical domains. The two protomers involved in the strand transfer reaction display a catalytically competent active site composed by three acidic residues (DDE), while the other two, which play a key role in the complex architecture, show catalytic pockets where the DDE residues are not properly positioned for cleavage. Quantification of in vivo transposition assays of mutants in key DNA binding residues, reveals that the lack of specificity generally decreases activity, but it could increase transposition in some cases. Our structure sheds light on the strand transfer reaction of the DDE DNA transposases and offers new insights into RNA-guided transposition in CAST systems.

biochemistry↗

bioRxiv2022

Structural remodelling of the carbon-phosphorus lyase machinery by a dual ABC ATPase

Phosphorus is an essential macronutrient for all microorganisms and can be extracted from the environment by several metabolic pathways. In Escherichia coli, the 14-cistron phn operon encoding the carbon-phosphorus (C-P) lyase enzymatic machinery allows for extraction of phosphorus from a wide range of phosphonates characterised by the highly stable C-P bond.1, 2 As part of a complex, multi-step pathway, the PhnJ subunit was proposed to cleave the C-P bond via a radical reaction, however, the details of the mechanism were not immediately compatible with the structure of the 220 kDa PhnGHIJ C-P lyase core complex, leaving a significant gap in our understanding of phosphonate breakdown in bacteria.3, 4 Here we show using single-particle cryogenic-electron microscopy that PhnJ mediates binding of a unique double dimer of ATP-binding cassette (ABC) proteins, PhnK and PhnL to the core complex. ATP hydrolysis by PhnK induces drastic structural remodelling leading to opening of the core and reconfiguration of a metal-binding site located at the interface between the PhnI and PhnJ subunits. Our results offer new insights into the mechanism underlying C-P lyase and uncover a hitherto unknown configuration of ABCs that have wide-ranging implications for our understanding of the role of this module in biological systems.

biochemistry↗

bioRxiv2021

Structure of the mini-RNA-guided endonuclease CRISPR-Cas{Phi}3

CRISPR-Cas{Phi} is a novel family of miniaturized RNA-guided endonucleases from phages 1,2. These novel ribonucleoproteins (RNPs) provide a compact scaffold gathering all key activities of a genome editing tool2. Here, we provide the first structural insight into Cas{Phi} singular DNA targeting and cleavage mechanism by determining the cryoEM structure of Cas{Phi}3 with the triple strand R-loop generated after DNA cleavage. The structure reveals the unique machinery for target unwinding to form the crRNA-DNA hybrid and cleaving the target DNA. The protospacer adjacent motif (PAM) is recognised by the target strand (T-strand) and non-target strand (NT-strand) PAM interacting domains (TPID and NPID). Unwinding occurs after insertion of the conserved 1 helix disrupting the dsDNA, thus facilitating the crRNA-DNA hybrid formation. The NT-strand is funnelled towards the RuvC catalytic site, while a long helix of TPID separates the displaced NT-strand and the crRNA-DNA hybrid avoiding DNA re-annealing. The crRNA-DNA hybrid is directed to the stop (STP) domain that splits the hybrid guiding the T-strand towards the RuvC active site. The conserved RuvC insertion of the Cas{Phi} family is extended along the hybrid, interacting with the phosphate backbone of the crRNA. A cluster of hydrophobic residues anchors the RuvC insertion in a cavity of the STP domain. The assembly of the hybrid promotes the shortening of the RuvC insertion, thus pulling the STP towards the RuvC active site to activate catalysis. These findings illustrate why Cas{Phi} unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. Site-directed mutagenesis in key residues support Cas{Phi}3 target DNA and non-specific ssDNA cutting mechanism. Our analysis provides new avenues to redesign the compact CRISPR-Cas{Phi} nucleases for genome editing.

biochemistry↗

bioRxiv2020

Structures of the Cmr-βComplex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas

Cmr-{beta} is a Type III-B CRISPR-Cas complex that upon target RNA recognition unleashes a multifaceted immune response against invading genetic elements, including ssDNA cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific ssRNA hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-{beta} unveiling how binding of the target RNA regulates the Cmr2 activities. CryoEM analysis revealed the unique subunit architecture of Cmr-{beta} and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5-handle in the crRNA and the 3-antitag of the target RNA, activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self vs. non-self recognition. These findings highlight the diverse defense strategies of Type III complexes.

biochemistry↗