The formation of many double-stranded DNA viruses, such as herpesviruses and bacteriophages, begins with the scaffolding-protein-mediated assembly of the procapsid. Subsequently, the procapsid undergoes extensive structural rearrangement and expansion to become the mature capsid. Bacteriophage P22 is an established model system used to study virus maturation. Here, we report the cryo-electron microscopy structures of procapsid, empty procapsid, empty mature capsid, and mature capsid of phage P22 at resolutions of 2.6 Å, 3.9 Å, 2.8 Å, and 3.0 Å, respectively. The structure of the procapsid allowed us to build an accurate model of the coat protein gp5 and the C-terminal region of the scaffolding protein gp8. In addition, interactions among the gp5 subunits responsible for procapsid assembly and stabilization were identified. Two C-terminal α-helices of gp8 were observed to interact with the coat protein in the procapsid. The amino acid interactions between gp5 and gp8 in the procapsid were consistent with the results of previous biochemical studies involving mutant proteins. Our structures reveal hydrogen bonds and salt bridges between the gp5 subunits in the procapsid and the conformational changes of the gp5 domains involved in the closure of the local sixfold opening and a thinner capsid shell during capsid maturation.

The Myoviridae phage tail is a common component of contractile injection systems (CISs), essential for exerting contractile function and facilitating membrane penetration of the inner tail tube. The near-atomic resolution structures of the Myoviridae tail have been extensively studied, but the dynamic conformational changes before and after contraction and the associated molecular mechanism are still unclear. Here, we present the extended and contracted intact tail-structures of Myoviridae phage P1 by cryo-EM. The ultra-long tail of P1, 2450 Å in length, consists of a neck, a tail terminator, 53 repeated tail sheath rings, 53 repeated tube rings, and a baseplate. The sheath of the contracted tail shrinks by approximately 55%, resulting in the separation of the inner rigid tail tube from the sheath. The extended and contracted tails were further resolved by local reconstruction at 3.3 Å and 3.9 Å resolutions, respectively, allowing us to build the atomic models of the tail terminator protein gp24, the tube protein BplB, and the sheath protein gp22 for the extended tail, and of the sheath protein gp22 for the contracted tail. Our atomic models reveal the complex interaction network in the ultra-long Myoviridae tail and the novel conformational changes of the tail sheath between extended and contracted states. Our structures provide insights into the contraction and stabilization mechanisms of the Myoviridae tail.

GP4, a new Ralstonia solanacearum phage, is a short-tailed phage. Few structures of Ralstonia solanacearum phages have been resolved to near-atomic resolution until now. Here, we present a 3.7 Å resolution structure of the GP4 head by cryo-electron microscopy (cryo-EM). The GP4 head contains 540 copies of major capsid protein (MCP) gp2 and 540 copies of cement protein (CP) gp1 arranged in an icosahedral shell with a triangulation number T = 9. The structures of gp2 and gp1 show a canonical HK97-like fold and an Ig-like fold, respectively. The trimeric CPs stick on the surface of the head along the quasi-threefold axis of the icosahedron generating a sandwiched three-layer electrostatic complementary potential, thereby enhancing the head stability. The assembly pattern of the GP4 head provides a platform for the further exploration of the interaction between Ralstonia solanacearum and corresponding phages.

Siphophages possess a long, flexible, and non-contractile tail that is responsible for host recognition, cell wall perforation, and genome delivery. Although the majority of structurally characterized siphophages target Gram-negative bacteria, those infecting Gram-positive bacteria remain elusive. Moreover, structural information concerning phage infection and genome release events in Gram-positive infecting siphophages is sparse. Here, we present a near-atomic resolution structure of Brochothrix thermosphacta bacteriophage NF5 determined by cryo-electron microscopy (cryo-EM). The structure comprises 11 proteins associated with the head, neck, tail tube, and baseplate, amounting to 643 polypeptides in total. Integration of cellular cryo-electron tomography (cryo-ET) showed the infection process of NF5, providing insights into the adsorption mechanism of siphophages targeting Gram-positive bacteria. Structural comparisons of baseplates from multiple siphophages targeting Gram-negative and Gram-positive bacteria reveal divergent compositional architectures and distinct assembly mechanisms. These disparities are evident in the domains across divergent baseplate proteins, suggesting evolutionary adaptations to host envelope architectures.

The continuous “arms race” between bacterial antiviral defense systems and phage anti-defense strategies drives evolutionary innovation. Previous study indicated that the defense against restriction (Dar) proteins DarA and Hdf in myophage P1 are associated with the head morphogenesis. However, the structural information for these proteins was lacking, and the mechanisms by which they mediate head morphogenesis and protect phage DNA against bacterial defense systems remained poorly understood. Using cryo-electron microscopy (cryo-EM), we resolved the entire structures of extended P1 and contracted P1 with partial DNA, with the latter lacking the baseplate, as well as the head structure of contracted P1 without DNA. We identified the structural proteins for the P1, including the head, connector complex, and baseplate, which exhibited conserved properties among the majority of myophages with a simple baseplate. Notably, 55 DarA-Hdf pairs are attached to the inner surface of head at each penton-hexon junction in the extended P1 and contracted P1 with partial DNA. The DarA and Hdf together form a complex that is tightly bound to the capsid and interacts with the DNA. However, these pairs are absent in the contracted P1 without DNA. Based on our three states of P1, we hypothesis that these extensive interactions among DarA, Hdf, DNA, and head play crucial roles in mediating capsid assembly, enhancing capsid stability, and protecting phage DNA. Our results provide a structural basis for further exploration of the mechanism by which Dar proteins function during phage assembly, infection and DNA ejection. This molecular mechanism may be conserved among P1-like phages.

Bacteriophages are increasingly considered viable alternatives to antibiotics for controlling aquaculture pathogens, yet the structural basis of siphophage infection in aquatic bacteria remains largely unexplored. Here, we present the near-atomic-resolution cryo-electron microscopy structure of Avs-1, a siphophage infecting the freshwater aquaculture pathogen Aeromonas veronii. The capsid reconstruction at 2.98 Å and spike structure at 3.58 Å reveal a virion architecture broadly conserved among lambda-like siphophages, including an HK97-fold capsid, a canonical portal–neck complex, and a tail tube composed of approximately 26 stacked hexameric rings. However, the distal tail tip exhibits pronounced specialization, assembling into a C3-symmetric tripod-like core formed by the distal tail protein, hub protein, insertion protein, and the C-terminus of the tape measure protein. The trimeric spike protein gp43 extends from this platform and adopts a β-helical scaffold with a structurally divergent distal domain (D4), highlighting the structural diversity of phage tail-tip architectures. Together, these results reveal how a largely conserved virion framework is combined with a diversified distal tail apparatus, expanding the structural repertoire of characterized siphophages and providing a framework for investigating the molecular basis of phage–host interactions.