Biology subjects

Zhou, Z. H.

Publications and source records attributed to Zhou, Z. H..

At least 19 recordsLinked to original sources

bioRxiv2026

Vault Packaging Dynamics: Cargo Loading Stabilizes MVP Conformation for High-Payload Cytokine Delivery in Cancer Therapy

Recombinant vaults are dynamic protein cages with a large interior volume that can encapsulate therapeutic cargo. Using integrative cryogenic electron microscopy (cryoEM), cryogenic electron tomography (cryoET) and charge-detection mass spectrometry (CDMS), we characterized recombinant empty and interleukin-2 (IL-2)-packaged human vaults produced in yeast to define assembly, packaging heterogeneity and conformational dynamics relevant to optimized cancer and immune therapeutics. CryoEM maps at 2.79 angstrom (packaged) and 3.16 angstrom (empty) reveal a D39-symmetric barrel built from 78 major vault protein (MVP) subunits each with an interaction domain (INT) that anchors a cargo on the inner surface at MVP repeats R3-R4. CDMS deconvolution of empty versus packaged particles yields two IL-2-INT vault populations centered near 100 and 400 molecules per vault, with a maximum of ~660 copies/vault (i.e., many more copies than the 78 available INT anchors), matching cryoET estimates of ~150-600. Both methods show that highly packaged vaults are enlarged and distorted. Empty particles are conformationally flexible, which promotes initial cargo entry; loading then stabilizes the barrel and condenses cargo, producing vaults that each contain many hundreds of IL-2 molecules. These data establish the structural basis of vault encapsulation and a quantitative framework for engineering vault-based delivery vehicles.

cell biology↗

bioRxiv2026

Using spIsoNet to address the preferred-orientation problem in cryoEM reconstructions

Cryogenic electron microscopy (cryoEM) is now routinely used for high-resolution structure determination of biological macromolecules. However, many biological specimens exhibit varying degrees of preferred orientation on cryoEM grids, resulting in uneven sampling of three-dimensional Fourier space. This orientation bias produces anisotropic reconstruction artifacts and, in severe cases, can exacerbate particle misalignment during iterative refinement, thereby limiting the success rate of near-atomic resolution cryoEM structure determination. This protocol provides a practical guide for applying spIsoNet, a self-supervised deep-learning method, to mitigate preferred-orientation issues in cryoEM reconstructions. We describe two complementary workflows: (1) map Anisotropy Correction to correct anisotropic artifacts of cryoEM maps and (2) particle Misalignment Correction, which integrates spIsoNet with RELION external reconstruction to improve particle-pose estimation. We demonstrate these workflows using two influenza hemagglutinin (HA) trimer datasets representing moderate and severe degrees of preferred-orientation bias. The protocol includes installation instructions, parameter-selection guidance, quality-control checkpoints and troubleshooting advice, and can typically be completed in [~]7 hours on a workstation equipped with four NVIDIA A100 GPUs. Together, these workflows provide step-by-step guidance for using the open-source spIsoNet software to mitigate the preferred-orientation problem directly from experimental data.

biophysics↗

bioRxiv2026

The tumour suppressor RBM5 activates the helicase DHX15 to regulate splicing

Pre-mRNA splicing determines the expressed proteome and is frequently dysregulated in cancer. The tumour-suppressor RBM5 controls an exon network regulating apoptosis, yet its molecular mechanism is elusive. Using in vivo spliceosome capture and cryogenic electron microscopy, we determined structures of precatalytic spliceosomes arrested by RBM5 immediately after U2 snRNP branchpoint recognition. Despite intron diversity, the U2-pre-mRNA duplex, branchpoint adenine, and downstream polypyrimidine tract are well-resolved. RBM5 binds the outer SF3B1 HEAT surface and performs dual functions: First, its helix-loop-helix motif and upstream zinc-finger domain sterically block tri-snRNP and Prp8 docking and prevent progression to pre-B and Bact complexes; Second, its G-patch activates DHX15 and places this DExH-box helicase on the pre-mRNA as it exits SF3B1, poised for branch helix unwinding. DHX15 binding to SF3B1 is facilitated by U2SURP/SR140, which engages SF3B1 near RBM5s helix-loop-helix. Functional assays confirm that disruption of the RBM5 interfaces with either DHX15 or SF3B1 inhibit exon repression. Mutations at these regulatory interfaces are common in cancer genomes and predicted to disrupt its regulation of apoptotic isoforms. Thus, RBM5 acts as a dual-action spliceosome gatekeeper that couples helicase activation with physical stalling to enforce tumour-suppressive alternative splicing programmes.

molecular biology↗

bioRxiv2026

A dramatic protein fold switch powers a bactericidal nanomachine

Fold switching, where a protein region interconverts between entirely distinct three-dimensional structures, is emerging as vital for certain protein functions. Here, we report a remarkable example in the F7 pyocin, a phage tail-like bactericidal nanomachine. Cryogenic electron microscopy and tomography reveal that a 163-residue segment of the central tail fiber undergoes a dramatic transition--from a trimeric -helical coiled-coil to a triangular {beta}-prism--upon binding to the bacterial cell surface. This massive fold switch remodels the tail tip, ejects the internal tape measure protein, and drives membrane puncture. Site-directed mutations that selectively destabilize the {beta}-prism conformation completely abolish bactericidal activity without impairing particle assembly, implying that the energy released during this transition powers penetration. AlphaFold-based analyses further predict similar large-scale coiled-coil to {beta}-prism switches in diverse non-contractile phage tails. This discovery reveals a sophisticated, ATP-independent strategy for microbial warfare and opens exciting possibilities for engineering next-generation bacteriocins to combat multidrug-resistant pathogens.

microbiology↗

bioRxiv2026

Snapshots of Internal Protein Crystal Architecture at the Nanoscale

Macromolecular crystallography has historically inferred models of internal crystal architecture from reciprocal-space measurements of Bragg reflections. Nevertheless, direct real-space visualization of crystallographic disorder remains elusive, particularly at the nanoscale. Using a 15-nanometer probe, here we apply both ambient-temperature and cryogenic four-dimensional scanning transmission electron microscopy (4D-STEM) to map the topography of coherently diffracting domains (CDDs) in lysozyme and myoglobin microcrystals at length scales 100x finer than conventional X-ray and electron beams. Virtual dark-field reconstructions from individual Bragg reflections reveal that each peak arises from spatially distinct subvolumes. Subdomain analysis demonstrates that these CDDs behave as orientationally monolithic units analogous to single mosaic blocks. Under sustained irradiation, protein CDDs undergo continuous rearrangements spanning several micrometers of internal movement. Furthermore, pinpoint high-dose "impact crater" experiments reveal delocalized radiolytic damage propagating hundreds of nanometers from primary irradiation sites, behavior similar to small-molecule crystals but amplified in both rate and magnitude. Together, these results establish macromolecular microcrystals as dynamic assemblies whose internal architecture continuously reorganizes during irradiation, laying the foundation for realistic models of mosaicity directly informed by both real-space and reciprocal-space observations.

biophysics↗

bioRxiv2025

IsoNet2 determines cellular structures at submolecular resolution without averaging

AbstractWe introduce IsoNet2, an end-to-end self-supervised deep-learning method that directly reconstructs high-quality 3D densities from cryogenic electron tomography. A unified network simultaneously performs denoising, contrast transfer function correction, and missing-wedge restoration, achieving [~]20 [A] resolution without averaging. A feature-rich GUI enables rapid, dataset-specific fine-tuning for end-users. IsoNet2 resolves domain organization in HIV capsid proteins, tRNA occupancy in individual ribosomes, and in situ architectures of mitochondrial respiration-related complexes, enabling atomic-level interpretation of cellular environments.

cell biology↗

bioRxiv2025

CryoDataBot: a pipeline to curate cryoEM datasets for AI-driven structural biology

Cryogenic electron microscopy (cryoEM) has revolutionized structural biology by enabling atomic-resolution visualization of biomacromolecules. To automate atomic model building from cryoEM maps, artificial intelligence (AI) methods have emerged as powerful tools. Although high-quality, task-specific datasets play a critical role in AI-based modeling, assembling such resources often requires considerable effort and domain expertise. We present CryoDataBot, an automated pipeline that addresses this gap. It streamlines data retrieval, preprocessing, and labeling, with fine-grained quality control and flexible customization, enabling efficient generation of robust datasets. CryoDataBots effectiveness is demonstrated through improved training efficiency in U-Net models and rapid, effective retraining of CryoREAD, a widely used RNA modeling tool. By simplifying the workflow and offering customizable quality control, CryoDataBot enables researchers to easily tailor dataset construction to the specific objectives of their models, while ensuring high data quality and reducing manual workload. This flexibility supports a wide range of applications in AI-driven structural biology.

molecular biology↗

bioRxiv2025

Toxoplasma IMC1 is a central component of the subpellicular network and plays critical roles in parasite morphology, replication, and infectivity

Toxoplasma gondii and related apicomplexan parasites utilize a unique membrane and cytoskeletal organelle called the inner membrane complex (IMC) for maintaining cell shape, motility, host cell invasion, and replication. The cytoskeleton portion of the organelle is a network of filaments composed of proteins called alveolins, whose precise functions and organization are poorly understood. Here we describe the function of the founding member of the Toxoplasma alveolins, IMC1, which we show is expressed and loaded onto forming daughter buds with IMC4, but later than the other key alveolins IMC3, IMC6, and IMC10. Disruption of IMC1 results in severe morphological defects that impact the integrity of the parasites cytoskeleton and disrupt invasion, replication, and egress. Loss of IMC1 in a less virulent type II strain results in a dramatic loss of infectivity and complete failure to form a chronic infection. We then use deletion analyses to dissect functional regions of the protein which reveals a key subregion of the alveolin domain that is sufficient for IMC targeting and also required for function. We then show that IMC1 interacts directly with IMC4 and the loss of IMC1 results in mislocalization of IMC4 specifically in forming daughter buds. This study thus reveals the critical role that IMC1 plays in forming and maintaining the architecture of the filamentous network of the IMC. SignificanceParasites in the phylum Apicomplexa maintain their intracellular lifestyle using specialized organelles that mediate the lytic cycle of host cell invasion, intracellular replication, and egress. One of these organelles is the inner membrane complex (IMC), which consists of membrane vesicles supported by a cytoskeletal meshwork formed from proteins called alveolins. This study focuses on the first identified alveolin IMC1 and determines its precise function via expression timing, gene knockout, deletion and mutagenesis, partner identification, and in vivo infection studies. We show that this protein is critical to the ultrastructure of the parasite which is important for every stage of its lytic cycle. We also identify key regions of the protein that are important for localization, function, and interaction with another key alveolin, IMC4.

microbiology↗

bioRxiv2025

CryoEM structure of the central apparatus of the Trypanosoma brucei flagellum

Axonemes are cylindrical bundles of microtubule filaments that typically follow a 9+n pattern (where n ranges from 0 to 4). However, variations exist across species and cell types, including architectures with fewer (e.g., 3+0, 6+0) or more than nine doublet microtubules (e.g., 9+9+0, 9+9+3), reflecting diverse structural adaptations of cilia and flagella in eukaryotes. Trypanosoma brucei, the causative agent of African trypanosomiasis, relies on its single 9+2 flagellum to navigate through environments within the mammalian host and insect vector. Central to the T. brucei flagellums function is a canonical central apparatus (CA), composed of two--C1 and C2-- singlet microtubules, which regulates flagellar beating and ensures efficient movement. Despite its crucial mechanoregulatory role in flagellar beating, the molecular structure and interactions governing T. brucei CA assembly and function remain poorly understood. In this study, we employed cryogenic electron microscopy (cryoEM) to uncover structural details of the T. brucei CA. We identified conserved and stably C1/C2-associated protein densities, including the armadillo repeat protein PF16, which serves as a structural scaffold critical for CA assembly and axonemal asymmetry. Our analysis also revealed pronounced molecular flexibility of the CA and uncovered T. brucei-specific densities, suggesting lineage-specific adaptations for parasite motility. These findings provide critical insights into the structural foundations of T. brucei motility. They also highlight potential therapeutic targets to disrupt the parasites ability to cause disease, offering new avenues for the treatment of African trypanosomiasis. Comparison of CAs in this canonical 9+2 axoneme and non-canonical 9+n axonemes offers general insights into the assembly and diverse functions of CAs across a wide range of species. SignificanceThe flagellum of Trypanosoma brucei, the parasite causing African trypanosomiasis, drives motility essential for host infection and disease transmission. Our cryogenic electron microscopy study reveals the molecular architecture of its central apparatus, identifying PF16 as a key scaffold that stabilizes axonemal asymmetry and imparts flexibility critical for flagellar beating. We uncover conserved proteins and T. brucei- specific adaptations, highlighting evolutionary divergence underlying the parasites distinctive motility. These structural results offer insights about how the central apparatus regulates T. bruceis bihelical motion and unveil potential therapeutic targets to disrupt flagellar function and combat African trypanosomiasis. Comparison among the central apparatus of canonical and non-canonical axonemes allow us to suggest broader principles underlying their central roles in ciliary functions across eukaryotes.

microbiology↗

bioRxiv2025

Discovery and CryoEM Structure of FPM13, a Periplasmic Metalloprotein Unique to Francisella

We report the cryoEM structure of the Francisella protein FTN_1118, identifying it as a novel 13 kDa periplasmic protein unique to the Francisella genus, which we now designate FPM13 (Francisella Periplasmic Metalloprotein, 13 kDa) based on its structural and biochemical properties. FPM13 was serendipitously identified during purification of Francisella type VI secretion system (T6SS) effector proteins, co-purifying with them. Its identity, initially unknown, was established using the novel cryoID method. The structure reveals a symmetrical, donut-shaped 18-mer with 9-fold dihedral symmetry, formed by two stacked nonamers head-to-head. It measures [~]8 nm both in height and in outer diameter, and has a 3.5 nm central channel. The complex features a double-layered wall with an inner {beta}-sheet core and an outer -helical shell. Each monomer adopts a compact fold comprising an N-terminus {beta}-strand, an -helix and two additional {beta} strands at the C-terminus. The assembly is stabilized by inter-ring loop interactions and hydrophobic and electrostatic contacts between neighboring subunits. Biochemical analyses, as shown by APEX-biotinylation and Triton X-114 phase partitioning, confirmed that FPM13 is a soluble periplasmic protein. ICP-MS demonstrated that FPM13 binds iron and copper. Deletion of FPM13 in Francisella novicida strains caused no growth defects in macrophages or mice but show increased copper sensitivity under iron-depleted conditions, suggesting a role for FPM13 in metal transport or detoxification.

microbiology↗

bioRxiv2025

Atomic Structure and Phospholipid Binding Properties of the Francisella Type VI Secretion System Effector Protein PdpC

Francisella tularensis, a gram-negative facultative intracellular pathogen, causes tularemia, a potentially fatal infection. The Francisella pathogenicity island (FPI) encodes a type VI secretion system (T6SS), critical for the bacteriums ability to replicate within host cells and cause disease. PdpC, an FPI-encoded T6SS effector protein, is essential for phagosomal escape, intracellular replication, and virulence in animals, yet its structure and function remain poorly understood. Here, we expressed PdpC recombinantly and determined the cryoEM structure of PdpC at 3.4 [A] resolution, revealing a monomeric 156 kDa protein with a seahorse-shaped architecture (80 [A] x 75 [A] x 120 [A]). PdpC comprises five domains: an N-terminal domain (NTD) forming a prominent lateral head lobe, a central body domain, a C-terminal tail domain, a small wedge domain at the groove between the body and tail, and an unmodeled "mouth" domain that completes the seahorse-like shape. Functional studies demonstrate that PdpC binds specific host cell phospholipids, including phosphatidylinositol-3-phosphate. These findings provide a foundation for elucidating PdpCs mechanism of action and developing strategies to prevent or treat tularemia.

microbiology↗

bioRxiv2025

Structure of a new capsid form and comparison with A-, B- and C-capsids clarify herpesvirus assembly and DNA translocation

Three capsid types have been recognized from the nuclei of herpesvirus-infected cells: empty A-capsids, scaffolding-containing B-capsids, and DNA-filled C-capsids. Despite progress in determining atomic structures of these capsids and extracellular virions in recent years, debate persists concerning the origins and temporal relationships among these capsids during capsid assembly and genome packaging. Here, we have imaged over 300,000 capsids of herpes simplex virus type 1 by cryogenic electron microscopy (cryoEM) and exhaustively classified them to characterize the structural heterogeneity of the DNA-translocating portal complex and their functional states. The resultant atomic structures reveal not only the expected A-, B-, and C-capsids, but also capsids with portal vertices similar to C-capsids but no resolvable genome in the capsid lumen, which we named D-capsids. The dodecameric dsDNA-translocating portal complex varies across these capsid types in their radial positions in icosahedral capsids and exhibits structural dynamics within each capsid type. In D-capsids, terminal DNA density exists in multiple conformations including one reminiscent to that in C-capsids, suggesting D-capsids are products of failed DNA retention. This interpretation is supported by varying amounts of DNA outside individual D-capsids and by correlation of capsid counts observed in situ of infected cell nuclei and those after purification. Additionally, an "anchoring" segment of the scaffold protein is resolved interacting with the portal baskets of A- and B-capsids but not D- and C-capsids. Taken together, our data indicate that A-capsids arise from failed DNA packaging and D-capsids from failed genome retention, clarifying the origins of empty capsids in herpesvirus assembly. SignificanceAs the prototypical herpesvirus, herpes simplex virus 1 (HSV-1) exhibits a global seroprevalence of 67% and approaching 90% in some localities. Herpesvirus infections can cause devastating cancers and birth defects, with HSV-1 infections leading to cold sores among the general population worldwide and blindness in developing nations. Here, we present atomic structures of the capsids sorted out from the nuclear isolates of HSV-1 infected cells, including the previously recognized A-, B-, and C-capsids, as well as the newly identified D-capsid. The structures show the details of protein-protein and protein-DNA interactions within each capsid type and the positional and interactional variability of the viral DNA-translocating portal vertex among these capsids. Importantly, our findings suggest that A-capsids are products of failed dsDNA packaging and D-capsids of failed genome retention. Together, the high-resolution 3D structures clarify the processes of genome packaging, maintenance, and ejection during capsid assembly, which are conserved across all herpesviruses.

microbiology↗

bioRxiv2025

Demonstration and structural basis of a therapeutic DNA aptamer for SARS-CoV-2 spike protein detection

At the onset of the COVID-19 pandemic, the absence of a rapid and highly specific diagnostic method for the SARS-CoV-2 virus led to significant delays in detection, adversely affecting clinical outcomes. This shortfall highlights the urgent need for adaptable, scalable, and reusable diagnostic technologies to improve future pandemic responses. To address this challenge, we developed a renewable electrochemical impedance biosensor device employing a synthetic nucleotide-based therapeutic aptamer (termed aptasensor) targeting the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). We demonstrate that our aptasensor can detect the Omicron BA.2 S protein within one hour and possesses concentration-dependent sensitivity at biologically relevant levels. Notably, the aptasensor is reusable after regeneration by a simple pH 2 buffer treatment. Aptamer binding to the S protein was confirmed by immunogold labeling and visualization by negative-stain electron microscopy. We used cryogenic electron microscopy (cryo-EM) to resolve high-resolution maps of the S protein in both the open and closed conformations and characterized aptamer binding to the up RBD in the open conformation. Taken together, these results establish the versatility and scalability of aptamer-based biosensors, presenting them as a potential transformative diagnostic platform for emerging pathogens. This combination of rapid detection, specificity, and renewable capabilities in a single diagnostic solution marks a significant advance in pandemic preparedness.

bioengineering↗

bioRxiv2025

Microplastics cross the murine intestine and induce inflammatory cell death after phagocytosis by human monocytes and neutrophils

Microplastics are contaminating the environment but also our food and drinking products. In a crucial study, microplastics have been detected in circulating human blood, urging the investigation of the effects of microplastics on human health. Here we aimed to determine the distribution of microplastics after oral exposure in mice and their interactions with and effects on mouse and human innate immune cells. We established that both 1 and 10m polystyrene (PS) particles penetrated the intestinal epithelium after oral administration and could be detected in blood and liver of mice after ten days of oral administration. Using intravital microscopy we captured the in vivo phagocytosis of 1m PS by mouse neutrophils in the liver. Pristine PS were barely phagocytosed by primary human phagocytes, however, 1 and 10{micro}m PS pre-incubated with plasma were readily phagocytosed by human neutrophils and monocytes. Plasma-coated 1 and 10m PS both increased human neutrophil and monocyte cell death but only after phagocytosis. Importantly, neutrophil cell death occurred a few hours after phagocytosing a single coated 10{micro}m PS while PS of 1{micro}m needed to be administered at a ratio of 27 particles per cell to induce significant neutrophil cell death. Neutrophil cell death upon microplastic exposure was characterized by extracellular DNA, which together with other released DAMPs can potentially trigger inflammation. Our findings suggest that microplastics could negatively impact the immune system and human health.

immunology↗

bioRxiv2025

Curvature generation and engineering principles from Shewanella oneidensis multi-flagellin flagellum

Motility driven by nanoscale flagella is vital to microbial survival and spread in fluid and structured environments. Absence of native flagellum structures, however, has limited our understanding of the mechanisms of microbial motility, hindering efforts to engineer microbe-based microbots for applications. Here, by cryogenic electron tomography (cryoET) and microscopy (cryoEM), we determined the structural basis of motility driven by the single flagellum anchored to one pole of Shewanella oneidensis MR-1 (S. oneidensis), an electrogenic bacterium commonly used in biotechnology. The structures of the curved flagellum, representing the conformation during motion, are captured, allowing delineation of molecular interactions among the subunits of its three components--filament, hook, and hook-filament junction. The structures of the filament, i.e., the propeller, reveal a varying composition of the flagellin isoforms FlaA and FlaB throughout the filament. Distinct inter-subunit interactions are identified at residues 129 and 134, which are the major determinants of functional differences in motility for the two isoforms. The hook--the universal joint--has a significantly larger curvature than that of the filament, despite both containing 11 curvature-defining conformers of their subunits. Transition between the propeller and universal joint is mediated by hook-filament junction, composed of 11 subunits of FlgK and FlgL, reconciling incompatibility between the filament and hook. Correlating these compositional and structural transitions with varying levels of curvature in flagellar segments reveals molecular mechanism enabling propulsive motility. Mechanistic understandings from S. oneidensis suggest engineering principles for nanoscale biomimetic systems. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/637127v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@17933forg.highwire.dtl.DTLVardef@1c44ab1org.highwire.dtl.DTLVardef@3d56dorg.highwire.dtl.DTLVardef@1b126b6_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Ethanolamine-induced assembly of microcompartments is required for Fusobacterium nucleatum virulence

Many bacteria metabolize ethanolamine as a nutrient source through cytoplasmic organelles named bacterial microcompartments (BMCs). Here we investigated the molecular assembly, regulation, and function of BMCs in Fusobacterium nucleatum - a Gram-negative oral pathobiont that is associated with adverse pregnancy outcomes. The F. nucleatum genome harbors a conserved ethanolamine utilization (eut) locus with 21 genes that encode several putative BMC shell proteins and a two-component signal transduction system (TCS), in addition to the enzymes for ethanolamine transport and catabolism. We show that the expression of most of these genes as well as BMC formation is highly increased in wild type fusobacteria when cultured in the presence of ethanolamine as a nutrient source. Deletion of the response regulator EutV eliminated this induction of eut mRNAs and BMCs, thus demonstrating that BMC formation is transcriptionally regulated by the TCS EutV-EutW in response to ethanolamine. Mass spectrometry of isolated BMCs unveiled the identity of the constituent proteins EutL, EutM1, EutM2, and EutN. Consistent with the role of these proteins in BMC assembly and metabolism, deletion of eutN, eutL/eutM1/eutM2, or eutL/eutM1/eutM2/eutN not only affected BMC formation, but also ethanolamine utilization, causing cell growth defects with ethanolamine as nutrient. BMCs also assembled in fusobacteria cultured with placental cells or the culture media, a process that is dependent on the BMC shell proteins. Significantly, we show that the eutN mutant is defective in inducing preterm birth in a mouse model. Together, these results establish that BMC-mediated metabolism of ethanolamine is critical for fusobacterial virulence. IMPORTANCEThe oral anaerobe Fusobacterium nucleatum can spread to distal internal organs, such as the colon and placenta, and thereby promote the development of colorectal cancer and induce preterm birth, respectively. Yet, how this opportunistic pathogen adapts to the various metabolically distinct host cellular niches remains poorly understood. We demonstrated here that this microbe assembles specialized metabolic organelles, termed bacterial microcompartments (BMCs), to utilize environmental ethanolamine (EA) as a key environmental nutrient source. The formation of F. nucleatum BMCs, containing BMC shell proteins EutLM1M2N, is controlled by a two-component system, EutV-EutW, responsive to EA. Significantly, this ability of F. nucleatum to form BMCs in response to EA is crucial for its pathogenicity evidenced by the fact that the genetic disruption of BMC formation reduces fusobacterial virulence in a mouse model of preterm birth.

microbiology↗

bioRxiv2024

Reconstruction and identification of the native PLP synthase complex from Methanosarcina acetivorans lysate

Many protein-protein interactions behave differently in biochemically purified forms as compared to their in vivo states. As such, determining native protein structures may elucidate structural states previously unknown for even well-characterized proteins. Here we apply the bottom-up structural proteomics method, cryoID, toward a model methanogenic archaeon. While they are keystone organisms in the global carbon cycle and active members of the human microbiome, there is a general lack of characterization of methanogen enzyme structure and function. Through the cryoID approach, we successfully reconstructed and identified the native Methanosarcina acetivorans pyridoxal 5-phosphate (PLP) synthase (PdxS) complex directly from cryogenic electron microscopy (cryoEM) images of fractionated cellular lysate. We found that the native PdxS complex exists as a homo-dodecamer of PdxS subunits, and the previously proposed supracomplex containing both the synthase (PdxS) and glutaminase (PdxT) was not observed in cellular lysate. Our structure shows that the native PdxS monomer fashions a single 8/8{beta} TIM-barrel domain, surrounded by seven additional helices to mediate solvent and interface contacts. A density is present at the active site in the cryoEM map and is interpreted as ribose 5-phosphate. In addition to being the first reconstruction of the PdxS enzyme from a heterogeneous cellular sample, our results reveal a departure from previously published archaeal PdxS crystal structures, lacking the 37 amino acid insertion present in these prior cases. This study demonstrates the potential of applying the cryoID workflow to capture native structural states at atomic resolution for archaeal systems, for which traditional biochemical sample preparation is nontrivial.

microbiology↗

bioRxiv2024

Native Doublet Microtubules from Trichomonas vaginalis Reveal Parasite-Specific Proteins as Potential Drug Targets

Doublet microtubules (DMTs) are flagellar components required for the protist Trichomonas vaginalis (Tv) to swim through the human genitourinary tract to cause trichomoniasis, the most common non-viral sexually transmitted disease. Lack of DMT structures has prevented structure-guided drug design to manage Tv infection. Here, we determined the cryo-EM structure of native Tv-DMTs, identifying 29 unique proteins, including 18 microtubule inner proteins and 9 microtubule outer proteins. While the A-tubule is simplistic compared to DMTs of other organisms, the B-tubule features specialized, parasite-specific proteins, like TvFAP40 and TvFAP35 that form filaments near the inner and outer junctions, respectively, to stabilize DMTs and enable Tv locomotion. Notably, a small molecule, assigned as IP6, is coordinated within a pocket of TvFAP40 and has characteristics of a drug molecule. This first atomic model of the Tv-DMT highlights the diversity of eukaryotic motility machinery and provides a structural framework to inform the rational design of therapeutics.

microbiology↗