Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex.

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman is a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact double-stranded DNA (dsDNA). When the HamAB complex detects DNA damage, HamB helicase activity activates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating "phantom" cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and enzymes from eukaryotes and archaea suggest deep functional symmetries with other important helicases across domains of life.

Time resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140 ms.

DdmDE eliminates plasmid invasion by DNA-guided DNA targeting.

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgos) and the DNA defense module DdmDE system. Through biochemical analysis, structural determination, and in vivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions.

Structure of TnsABCD transpososome reveals mechanisms of targeted DNA transposition.

Tn7-like transposons are characterized by their ability to insert specifically into host chromosomes. Recognition of the attachment (att) site by TnsD recruits the TnsABC proteins to form the transpososome and facilitate transposition. Although this pathway is well established, atomic-level structural insights of this process remain largely elusive. Here, we present the cryo-electron microscopy (cryo-EM) structures of the TnsC-TnsD-att DNA complex and the TnsABCD transpososome from the Tn7-like transposon in Peltigera membranacea cyanobiont 210A, a type I-B CRISPR-associated transposon. Our structures reveal a striking bending of the att DNA, featured by the intercalation of an arginine side chain of TnsD into a CC/GG dinucleotide step. The TnsABCD transpososome structure reveals TnsA-TnsB interactions and demonstrates that TnsC not only recruits TnsAB but also directly participates in the transpososome assembly. These findings provide mechanistic insights into targeted DNA insertion by Tn7-like transposons, with implications for improving the precision and efficiency of their genome-editing applications.

Viral DNA polymerase structures reveal mechanisms of antiviral drug resistance.

DNA polymerases are important drug targets, and many structural studies have captured them in distinct conformations. However, a detailed understanding of the impact of polymerase conformational dynamics on drug resistance is lacking. We determined cryoelectron microscopy (cryo-EM) structures of DNA-bound herpes simplex virus polymerase holoenzyme in multiple conformations and interacting with antivirals in clinical use. These structures reveal how the catalytic subunit Pol and the processivity factor UL42 bind DNA to promote processive DNA synthesis. Unexpectedly, in the absence of an incoming nucleotide, we observed Pol in multiple conformations with the closed state sampled by the fingers domain. Drug-bound structures reveal how antivirals may selectively bind enzymes that more readily adopt the closed conformation. Molecular dynamics simulations and the cryo-EM structure of a drug-resistant mutant indicate that some resistance mutations modulate conformational dynamics rather than directly impacting drug binding, thus clarifying mechanisms that drive drug selectivity.

Structure-based design of non-hypertrophic apelin receptor modulator.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

Structure of the plant plastid-encoded RNA polymerase.

Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined. Here, we present cryoelectron microscopy (cryo-EM) structures of native 21-subunit PEP and a PEP transcription elongation complex from white mustard (Sinapis alba). We identify that PAPs encase the core polymerase, forming extensive interactions that likely promote complex assembly and stability. During elongation, PAPs interact with DNA downstream of the transcription bubble and with the nascent mRNA. The models reveal details of the superoxide dismutase, lysine methyltransferase, thioredoxin, and amino acid ligase enzymes that are subunits of PEP. Collectively, these data provide a foundation for the mechanistic understanding of chloroplast transcription and its role in plant growth and adaptation.

Molecular basis of global promoter sensing and nucleosome capture by the SWR1 chromatin remodeler.

The SWR1 chromatin remodeling complex is recruited to +1 nucleosomes downstream of transcription start sites of eukaryotic promoters, where it exchanges histone H2A for the specialized variant H2A.Z. Here, we use cryoelectron microscopy (cryo-EM) to resolve the structural basis of the SWR1 interaction with free DNA, revealing a distinct open conformation of the Swr1 ATPase that enables sliding from accessible DNA to nucleosomes. A complete structural model of the SWR1-nucleosome complex illustrates critical roles for Swc2 and Swc3 subunits in oriented nucleosome engagement by SWR1. Moreover, an extended DNA-binding α helix within the Swc3 subunit enables sensing of nucleosome linker length and is essential for SWR1-promoter-specific recruitment and activity. The previously unresolved N-SWR1 subcomplex forms a flexible extended structure, enabling multivalent recognition of acetylated histone tails by reader domains to further direct SWR1 toward the +1 nucleosome. Altogether, our findings provide a generalizable mechanism for promoter-specific targeting of chromatin and transcription complexes.

Structural insights into the chloroplast protein import in land plants.

Chloroplast proteins are imported via the translocon at the outer chloroplast membrane (TOC)-translocon at the inner chloroplast membrane (TIC) supercomplex, driven by an ATPase motor. The Ycf2-FtsHi complex has been identified as the chloroplast import motor. However, its assembly and cooperation with the TIC complex during preprotein translocation remain unclear. Here, we present the structures of the Ycf2-FtsHi and TIC complexes from Arabidopsis and an ultracomplex formed between them from Pisum. The Ycf2-FtsHi structure reveals a heterohexameric AAA+ ATPase motor module with characteristic features. Four previously uncharacterized components of Ycf2-FtsHi were identified, which aid in complex assembly and anchoring of the motor module at a tilted angle relative to the membrane. When considering the structures of the TIC complex and the TIC-Ycf2-FtsHi ultracomplex together, it becomes evident that the tilted motor module of Ycf2-FtsHi enables its close contact with the TIC complex, thereby facilitating efficient preprotein translocation. Our study provides valuable structural insights into the chloroplast protein import process in land plants.

Conservation and specialization of the Ycf2-FtsHi chloroplast protein import motor in green algae.

The protein import motor in chloroplasts plays a pivotal role in their biogenesis and homeostasis by driving the translocation of preproteins into chloroplasts. While the Ycf2-FtsHi complex serves as the import motor in land plants, its evolutionary conservation, specialization, and mechanisms across photosynthetic organisms are largely unexplored. Here, we isolated and determined the cryogenic electron microscopy (cryo-EM) structures of the native Ycf2-FtsHi complex from Chlamydomonas reinhardtii, uncovering a complex composed of up to 19 subunits, including multiple green-algae-specific components. The heterohexameric AAA+ ATPase motor module is tilted, potentially facilitating preprotein handover from the translocon at the inner chloroplast membrane (TIC) complex. Preprotein interacts with Ycf2-FtsHi and enhances its ATPase activity in vitro. Integrating Ycf2-FtsHi and translocon at the outer chloroplast membrane (TOC)-TIC supercomplex structures reveals insights into their physical and functional interplay during preprotein translocation. By comparing these findings with those from land plants, our study establishes a structural foundation for understanding the assembly, function, evolutionary conservation, and diversity of chloroplast protein import motors.

Assembly and activation of EBV latent membrane protein 1.

Latent membrane protein 1 (LMP1) is the primary oncoprotein of Epstein-Barr virus (EBV) and plays versatile roles in the EBV life cycle and pathogenesis. Despite decades of extensive research, the molecular basis for LMP1 folding, assembly, and activation remains unclear. Here, we report cryo-electron microscopy structures of LMP1 in two unexpected assemblies: a symmetric homodimer and a higher-order filamentous oligomer. LMP1 adopts a non-canonical and unpredicted fold that supports the formation of a stable homodimer through tight and antiparallel intermolecular packing. LMP1 dimers further assemble side-by-side into higher-order filamentous oligomers, thereby allowing the accumulation and specific organization of the flexible cytoplasmic tails for efficient recruitment of downstream factors. Super-resolution microscopy and cellular functional assays demonstrate that mutations at both dimeric and oligomeric interfaces disrupt LMP1 higher-order assembly and block multiple LMP1-mediated signaling pathways. Our research provides a framework for understanding the mechanism of LMP1 and for developing potential therapies targeting EBV-associated diseases.

Cryo-EM-based discovery of a pathogenic parvovirus causing epidemic mortality by black wasting disease in farmed beetles.

We use cryoelectron microscopy (cryo-EM) as a sequence- and culture-independent diagnostic tool to identify the etiological agent of an agricultural pandemic. For the past 4 years, American insect-rearing facilities have experienced a distinctive larval pathology and colony collapse of farmed Zophobas morio (superworm). By means of cryo-EM, we discovered the causative agent: a densovirus that we named Zophobas morio black wasting virus (ZmBWV). We confirmed the etiology of disease by fulfilling Koch's postulates and characterizing strains from across the United States. ZmBWV is a member of the family Parvoviridae with a 5,542 nt genome, and we describe intersubunit interactions explaining its expanded internal volume relative to human parvoviruses. Cryo-EM structures at resolutions up to 2.1 Å revealed single-strand DNA (ssDNA) ordering at the capsid inner surface pinned by base-binding pockets in the capsid inner surface. Also, we demonstrated the prophylactic potential of non-pathogenic strains to provide cross-protection in vivo.

Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies.

Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

Diatom pyrenoids are encased in a protein shell that enables efficient CO2 fixation.

Pyrenoids are subcompartments of algal chloroplasts that increase the efficiency of Rubisco-driven CO2 fixation. Diatoms fix up to 20% of global CO2, but their pyrenoids remain poorly characterized. Here, we used in vivo photo-crosslinking to identify pyrenoid shell (PyShell) proteins, which we localized to the pyrenoid periphery of model pennate and centric diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana. In situ cryo-electron tomography revealed that pyrenoids of both diatom species are encased in a lattice-like protein sheath. Single-particle cryo-EM yielded a 2.4-Å-resolution structure of an in vitro TpPyShell1 lattice, which showed how protein subunits interlock. T. pseudonana TpPyShell1/2 knockout mutants had no PyShell sheath, altered pyrenoid morphology, and a high-CO2 requiring phenotype, with reduced photosynthetic efficiency and impaired growth under standard atmospheric conditions. The structure and function of the diatom PyShell provide a molecular view of how CO2 is assimilated in the ocean, a critical ecosystem undergoing rapid change.

Potent and broad HIV-1 neutralization in fusion peptide-primed SHIV-infected macaques.

An antibody-based HIV-1 vaccine will require the induction of potent cross-reactive HIV-1-neutralizing responses. To demonstrate feasibility toward this goal, we combined vaccination targeting the fusion-peptide site of vulnerability with infection by simian-human immunodeficiency virus (SHIV). In four macaques with vaccine-induced neutralizing responses, SHIV infection boosted plasma neutralization to 45%-77% breadth (geometric mean 50% inhibitory dilution [ID50] ∼100) on a 208-strain panel. Molecular dissection of these responses by antibody isolation and cryo-electron microscopy (cryo-EM) structure determination revealed 15 of 16 antibody lineages with cross-clade neutralization to be directed toward the fusion-peptide site of vulnerability. In each macaque, isolated antibodies from memory B cells recapitulated the plasma-neutralizing response, with fusion-peptide-binding antibodies reaching breadths of 40%-60% (50% inhibitory concentration [IC50] < 50 µg/mL) and total lineage-concentrations estimates of 50-200 µg/mL. Longitudinal mapping indicated that these responses arose prior to SHIV infection. Collectively, these results provide in vivo molecular examples for one to a few B cell lineages affording potent, broadly neutralizing plasma responses.

The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity.

Type III CRISPR systems provide immunity against genetic invaders through the production of cyclic oligo-adenylate (cAn) molecules that activate effector proteins that contain CRISPR-associated Rossman fold (CARF) domains. Here, we characterized the function and structure of an effector in which the CARF domain is fused to an adenosine deaminase domain, CRISPR-associated adenosine deaminase 1 (Cad1). We show that upon binding of cA4 or cA6 to its CARF domain, Cad1 converts ATP to ITP, both in vivo and in vitro. Cryoelectron microscopy (cryo-EM) structural studies on full-length Cad1 reveal an hexameric assembly composed of a trimer of dimers, with bound ATP at inter-domain sites required for activity and ATP/ITP within deaminase active sites. Upon synthesis of cAn during phage infection, Cad1 activation leads to a growth arrest of the host that prevents viral propagation. Our findings reveal that CRISPR-Cas systems employ a wide range of molecular mechanisms beyond nucleic acid degradation to provide adaptive immunity in prokaryotes.

RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells.

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.

POT1 recruits and regulates CST-Polα/primase at human telomeres.

Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.

Integrated cryoEM structure of a spumaretrovirus reveals cross-kingdom evolutionary relationships and the molecular basis for assembly and virus entry.

Foamy viruses (FVs) are an ancient lineage of retroviruses, with an evolutionary history spanning over 450 million years. Vector systems based on Prototype Foamy Virus (PFV) are promising candidates for gene and oncolytic therapies. Structural studies of PFV contribute to the understanding of the mechanisms of FV replication, cell entry and infection, and retroviral evolution. Here we combine cryoEM and cryoET to determine high-resolution in situ structures of the PFV icosahedral capsid (CA) and envelope glycoprotein (Env), including its type III transmembrane anchor and membrane-proximal external region (MPER), and show how they are organized in an integrated structure of assembled PFV particles. The atomic models reveal an ancient retroviral capsid architecture and an unexpected relationship between Env and other class 1 fusion proteins of the Mononegavirales. Our results represent the de novo structure determination of an assembled retrovirus particle.