The type I-FHNH CRISPR-Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous DNA cleavage without accessory nucleases. Using cryo-EM, we determined high-resolution structures of the effector complex in three catalytic states-precatalytic, NTS-cleaved, and post-catalytic-revealing a dynamic trajectory of the HNH domain through inward, middle, and outward conformations. Biochemical assays demonstrated that the complex cleaves the nontarget strand (NTS) prior to the target strand (TS), consistent with a sequential cleavage mechanism similar to Cas12 effectors but notably lacking trans-cleavage activity on single-stranded DNA. Structural comparisons confirmed a minimal PAM requirement (5'-CN) and a constrained HNH catalytic site poised for precise strand scission. We engineered a ΔLinker variant of Cas8 that repositions the HNH domain, selectively abolishing TS cleavage and converting the system into a programmable NTS-specific nickase. Importantly, we validated the functionality of both wild-type and mutant complexes in human cells. While the wild-type system induced indels and base substitutions, the ΔLinker variant triggered targeted single-strand nicks without double-stranded breaks. Together, our work establishes type I-FHNH as a compact and precise genome editing platform with in vivo efficacy.

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 and have been engineered to deliver diverse protein cargoes into non-natively-targeted organisms. Despite the structural insights into several CISs, including one PVC from P. asymbiotica, 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. This particle 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 and the baseplate of the contracted particle, yielding insight into the contraction mechanism. This study provides structural details of the extended and contracted states of the PlPVC1 particle and supports the model in which contraction is triggered. Furthermore, it facilitates the comparison of PlPVC1 with other CISs and expands the scope of engineering opportunities for future biomedical and biotechnological applications.

Bacteriophages use receptor-binding proteins (RBPs) to adhere to bacterial hosts, yet their sequence and structural diversity remain poorly understood. Tail fibers, a major class of RBPs, are elongated and flexible trimeric proteins, making their full-length structures difficult to resolve experimentally. Advances in deep learning-based protein structure prediction, such as AlphaFold2-multimer (AF2M) and ESMFold, provide opportunities for studying these challenging proteins. Here, we introduce RBPseg, a method that combines monomeric ESMFold predictions with a structural-based domain identification approach, to divide tail fiber sequences into manageable fractions for high-confidence modeling with AF2M. Using this approach, we generated complete tail fiber models, validated by single-particle cryo-electron microscopy of five fibers from three phages. A structural classification of 67 fibers identified 16 distinct classes and 89 domains, revealing patterns of modularity, convergence, divergence, and domain swapping. Our findings suggest that these structural classes represent at least 24% of the known tail fiber universe, providing key insights into their evolution and functionality.

CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.

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. ### Competing Interest Statement The authors have declared no competing interest.

In Escherichia coli , the 14-cistron phn operon encoding carbon-phosphorus lyase allows for utilisation of phosphorus from a wide range of stable phosphonate compounds containing a C-P bond. As part of a complex, multi-step pathway, the PhnJ subunit was shown to cleave the C-P bond via a radical mechanism, however, the details of the reaction could not immediately be reconciled with the crystal structure of a 220 kDa PhnGHIJ C-P lyase core complex, leaving a significant gap in our understanding of phosphonate breakdown in bacteria. Here, we show using single-particle cryogenic electron microscopy that PhnJ mediates binding of a double dimer of the ATP-binding cassette proteins, PhnK and PhnL, to the core complex. ATP hydrolysis induces drastic structural remodelling leading to opening of the core complex and reconfiguration of a metal-binding and putative active site located at the interface between the PhnI and PhnJ subunits.

Phosphorus (P) is an essential macronutrient that can be extracted from the environment by several metabolic pathways. In E. coli, phosphate deprivation activates the the 14-cistron phn operon that encodes carbon-phosphorus (C-P) lyase. C-P lyase confers the ability to extract phosphorus from a wide range of phosphonate compounds containing the chemically highly stable C-P bond. Phosphonates are widespread in nature and due to their similarity to phosphate esters, have important applications in agriculture and biomedicine, as pesticides (glyphosate, RoundUp) and antibiotics, respectively. C-P lyase catalyses a complex multi-step pathway that directly depends on 10 of the encoded proteins (PhnGHIJKLMNOP). Of these, the PhnJ subunit was shown to be responsible for cleavage of the C-P bond via a strict anaerobic glycyl radical mechanism that requires an iron-sulphur (Fe S ) cluster and S-adenosyl methionine (SAM) for radical activation. Surprisingly, however, this mechanism was not immediately compatible with the crystal structure of a PhnGHIJ C-P lyase core complex, which placed key residues at a significant distance, thus leaving a large gap in our understanding of the mechanism of phosphonate breakdown. The phn operon also encodes two ATP-binding cassette (ABC) proteins with homology to the nucleotide-binding domains of ABC transporters, PhnK and PhnL, for which no function has been assigned. Here, we present four high-resolution (~2 Å), single-particle cryo-electron microscopy structures of 300+ kDa C-P lyase complexes in several key, functional states. Together, these structures reveal how the PhnGHIJ core complex interacts with a unique double dimer of PhnK and PhnL subunits, in which PhnK, in a tight ATP-bound conformation, bridges the core complex and PhnL. We also show using additional structures determined under ATP turnover conditions, that ATP hydrolysis in PhnK induces a dramatic remodelling of the core complex, leading to opening and large-scale movement of several subunits. Moreover, we show that ATP hydrolysis and binding of both ABC subunits is required for growth of E. coli on phosphonate. Finally, we demonstrate that C-P lyase in the closed state binds substrate at a metal-binding site located at the interface between the PhnI and PhnJ subunits, and that ATP hydrolysis leads to a reorganisation of this active site in a way likely contributes to substrate/product exchange. In summary, we provide several novel insights into the elusive process of phosphonate breakdown by C-P lyase in microorganisms and represent a solid structural basis for understanding the catalytic process. Our structural data also uncover a hitherto unknown configuration of ABCs in which two ABC dimers directly interact with each other that have broad implications for our understanding of the role of this module in biological systems. 1. Metcalf, W. W. & Wanner, B. L. Gene 129, 27-32, (1993). 2. Kamat, S. S., Williams, H. J., Dangott, L. J., Chakrabarti, M. & Raushel, F. M. Nature 497, 132-136, (2013). 3. Seweryn, P. et al. Nature 525, 68-72, (2015).

CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opted CRISPR-Cas systems for RNA-guided transposition. Here we present the 2.4 Å cryo-EM structure of the Scytonema hofmannii (sh) TnsB transposase from Type V-K CAST, bound to the strand transfer DNA. The strand transfer complex displays an intertwined pseudo-symmetrical architecture. Two protomers involved in strand transfer display a catalytically competent active site composed by DDE residues, while other two, which play a key structural role, show active sites where the catalytic residues are not properly positioned for phosphodiester hydrolysis. Transposon end recognition is accomplished by the NTD1/2 helical domains. A singular in trans association of NTD1 domains of the catalytically competent subunits with the inactive DDE domains reinforces the assembly. Collectively, the structural features suggest that catalysis is coupled to protein-DNA assembly to secure proper DNA integration. DNA binding residue mutants reveal that lack of specificity decreases activity, but it could increase transposition in some cases. Our structure sheds light on the strand transfer reaction of DDE transposases and offers new insights into CAST transposition.

CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.

CRISPR-CasΦ is a novel family of miniaturized RNA-guided endonucleases from phages [1][1],[2][2]. These novel ribonucleoproteins (RNPs) provide a compact scaffold gathering all key activities of a genome editing tool[2][2]. Here, we provide the first structural insight into CasΦ singular DNA targeting and cleavage mechanism by determining the cryoEM structure of CasΦ3 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 α1 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 CasΦ 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 CasΦ unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. Site-directed mutagenesis in key residues support CasΦ3 target DNA and non-specific ssDNA cutting mechanism. Our analysis provides new avenues to redesign the compact CRISPR-CasΦ nucleases for genome editing. ### Competing Interest Statement Guillermo Montoya and Stefano Stella declare that they are co-founders of Twelve Bio. A patent application has been filed relating to this work. [1]: #ref-1 [2]: #ref-2

CRISPR loci and CRISPR-associated (Cas) genes encode an adaptive immune system that protects many bacterial and almost all archaea against invasive genetic elements from bacteriophages and plasmids. Several classes of CRISPR systems have been characterized, of which the type III CRISPR systems exhibit the most unique functions. Members of type III cleave both RNA and DNA not only through their corresponding effector complexes but also by CRISPR-Cas associated proteins activated by second messengers produced by those effector complexes. Furthermore, the recent discovery of second messenger degrading proteins called ring nucleases adds an extra regulatory layer to fine-tune these immunity systems. Here, we review the defense mechanisms that govern type III CRISPR interference immunity systems focusing on the structural information available.

Cmr-β 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-β unveiling how binding of the target RNA regulates the Cmr2 activities. CryoEM analysis revealed the unique subunit architecture of Cmr-β 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.

Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA 4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA 4 binding site and a ssRNA catalytic pocket. cOA 4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA 4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5′-C-C-3′. The degradation of cOA 4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.

Bacteria have evolved advanced strategies for surviving during nutritional stress, including expression of specialized enzyme systems that allow them to grow on unusual nutrient sources. Inorganic phosphate (Pi ) is limiting in most ecosystems, hence organisms have developed a sophisticated, enzymatic machinery known as carbon-phosphorus (C-P) lyase, allowing them to extract phosphate from a wide range of phosphonate compounds. These are characterized by a stable covalent bond between carbon and phosphorus making them very hard to break down. Despite the challenges involved in both synthesizing and catabolizing phosphonates, they are widespread in nature. The enzymes required for the bacterial C-P lyase pathway have been identified and for the most part structurally characterized. Nevertheless, the mechanistic principles governing breakdown of phosphonate compounds remain enigmatic. In this review, an overview of the C-P lyase pathway is provided and structural aspects of the involved enzyme complexes are discussed with a special emphasis on the role of ATP-binding cassette (ABC) proteins.

RNA decay is an important process that is essential for controlling the abundance, quality and maturation of transcripts. In eukaryotes, RNA decay in the 3'-5' direction is carried out by the exosome, an RNA-degradation machine that is conserved from yeast to humans. A range of cofactors stimulate the enzymatic activity of the exosome and serve as adapters for the many RNA substrates. In human cells, the exosome associates with the heterotrimeric nuclear exosome targeting (NEXT) complex consisting of the DExH-box helicase hMTR4, the zinc-finger protein hZCCHC8 and the RRM-type protein hRBM7. Here, the 2.5 Å resolution crystal structure of the RRM domain of human RBM7 is reported. Molecular replacement using a previously determined solution structure of RBM7 was unsuccessful. Instead, RBM8 and CBP20 RRM-domain crystal structures were used to successfully determine the RBM7 structure by molecular replacement. The structure reveals a ring-shaped pentameric assembly, which is most likely a consequence of crystal packing.

Activation of toxin–antitoxin (TA) systems provides an important mechanism for bacteria to adapt to challenging and ever changing environmental conditions. Known TA systems are classified into five families based on the mechanisms of antitoxin inhibition and toxin activity. For type II TA systems, the toxin is inactivated in exponentially growing cells by tightly binding its antitoxin partner protein, which also serves to regulate cellular levels of the complex through transcriptional auto-repression. During cellular stress, however, the antitoxin is degraded thus freeing the toxin, which is then able to regulate central cellular processes, primarily protein translation to adjust cell growth to the new conditions. In this review, we focus on the type II TA pairs that regulate protein translation through cleavage of ribosomal, transfer, or messenger RNA.

Activation of toxin–antitoxin (TA) systems provides an important mechanism for bacteria to adapt to challenging and ever changing environmental conditions. Known TA systems are classified into five families based on the mechanisms of antitoxin inhibition and toxin activity. For type II TA systems, the toxin is inactivated in exponentially growing cells by tightly binding its antitoxin partner protein, which also serves to regulate cellular levels of the complex through transcriptional auto-repression. During cellular stress, however, the antitoxin is degraded thus freeing the toxin, which is then able to regulate central cellular processes, primarily protein translation to adjust cell growth to the new conditions. In this review, we focus on the type II TA pairs that regulate protein translation through cleavage of ribosomal, transfer, or messenger RNA.

The bacterial relBE locus encodes a toxin-antitoxin complex in which the toxin, RelE, is capable of cleaving mRNA in the ribosomal A site cotranslationally. The antitoxin, RelB, both binds and inhibits RelE, and regulates transcription through operator binding and conditional cooperativity controlled by RelE. Here, we present the crystal structure of the intact Escherichia coli RelB2E2 complex at 2.8 Å resolution, comprising both the RelB-inhibited RelE and the RelB dimerization domain that binds DNA. RelE and RelB associate into a V-shaped heterotetrameric complex with the ribbon-helix-helix (RHH) dimerization domain at the apex. Our structure supports a model in which relO is optimally bound by two adjacent RelB2E heterotrimeric units, and is not compatible with concomitant binding of two RelB2E2 heterotetramers. The results thus provide a firm basis for understanding the model of conditional cooperativity at the molecular level.