Abstract Cryogenic electron tomography (cryoET) allows visualization of cellular structures in situ. However, anisotropic resolution arising from the intrinsic “missing-wedge” problem has presented major challenges and interpretation tomograms. Here, we have developed IsoNet, a deep learning-based software package that iteratively reconstructs missing-wedge information increases signal-to-noise ratio, using knowledge learned raw Without need for sub-tomogram averaging, IsoNet generates...

The 96-nm axonemal repeat includes dynein motors and accessory structures as the foundation for motility of eukaryotic flagella cilia. However, high-resolution 3D axoneme are unavailable organisms among Excavates, which include pathogens medical economic importance. Here we report cryo electron tomography from Trypanosoma brucei, a protozoan parasite in Excavate lineage that causes African trypanosomiasis. We examined bloodstream procyclic life cycle stages, knockdown lacking DRC11/CMF22...

Abstract Cryogenic electron tomography (cryoET) allows visualization of cellular structures in situ . However, anisotropic resolution arising from the intrinsic “missing-wedge” problem has presented major challenges and interpretation tomograms. Here, we have developed IsoNet, a deep learning-based software package that iteratively reconstructs missing-wedge information increases signal-to-noise ratio, using knowledge learned raw Without need for sub-tomogram averaging, Isonet generates...

The flagellum of Trypanosoma brucei drives the parasite’s characteristic screw-like motion and is essential for its replication, transmission, pathogenesis. However, molecular details this process remain unclear. Here, we present high-resolution (up to 2.8 angstrom) cryo–electron microscopy structures T. flagellar doublet microtubules (DMTs). Integrated modeling identified 154 different axonemal proteins inside outside DMT and, together with genetic proteomic interrogation, revealed...

Motility of pathogenic protozoa depends on flagella (synonymous with cilia) axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules. Microtubule inner proteins (MIPs) within DMTs influence axoneme stability motility provide lineage-specific adaptations, but individual MIP functions assembly mechanisms are mostly unknown. Here, we show in the sleeping sickness parasite Trypanosoma brucei, that FAP106, a conserved at DMT junction, is required for trypanosome as...

Cryogenic electron tomography (cryoET) is capable of determining in situ biological structures molecular complexes at near atomic resolution by averaging half a million subtomograms. While abundant complexes/particles are often clustered arrays, precisely locating and seamlessly such particles across many tomograms present major challenges. Here, we developed TomoNet, software package with modern graphical user interface to carry out the entire pipeline cryoET subtomogram achieve high...

Abstract Cryogenic electron tomography (cryoET) is capable of determining in situ biological structures molecular complexes at near-atomic resolution by averaging half a million subtomograms. While abundant complexes/particles are often clustered arrays, precisely locating and seamlessly such particles across many tomograms present major challenges. Here, we developed TomoNet, software package with modern graphical user interface to carry out the entire pipeline cryoET subtomogram achieve...

Eukaryotic flagella (synonymous with cilia) rely on a microtubule-based axoneme, together accessory filaments to carryout motility and signaling functions. While axoneme structures are well characterized, 3D ultrastructure of their interface mostly unknown, presenting critical gap in understanding structural foundations eukaryotic flagella. In the flagellum protozoan parasite Trypanosoma brucei (T. brucei), is accompanied by paraflagellar rod (PFR) that supports non-planar necessary for...

Abstract Certain archaeal cells possess external proteinaceous sheath, whose structure and organization are both unknown. By cellular cryogenic electron tomography (cryoET), here we have determined sheath of the prototypical archaeon, Methanospirillum hungatei . Fitting Alphafold-predicted model protein (SH) monomer into 7.9 Å-resolution reveals that cylinder consists axially stacked β-hoops, each which is comprised two to six 400 nm-diameter rings β-strand arches (β-rings). With...

Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to cytoplasm. This initial step of nuclear egress-budding at inner membrane-is coordinated by complex (NEC). Composed viral proteins UL31 and UL34, NEC deforms membrane around capsid as latter buds into perinuclear space. oligomerization a hexagonal membrane-bound lattice for budding because mutants designed perturb interfaces reduce its ability. Previously, we identified...

Abstract Background Resolution estimation is the main evaluation criteria for reconstruction of macromolecular 3D structure in field cryoelectron microscopy (cryo-EM). At present, there are many methods to evaluate resolution reconstructed structures from Single Particle Analysis (SPA) cryo-EM and subtomogram averaging (SA) electron cryotomography (cryo-ET). As global methods, they measure as a whole, but inaccurate detecting subtle local changes reconstruction. In order detect SPA SA, few...

Rabies virus (RABV) is among the first recognized viruses of public health concern and has historically contributed to development viral vaccines. Despite these significances, three-dimensional structure RABV virion remains unknown due challenges in isolating structurally homogenous samples sufficient quantities needed for structural investigation. Here, by combining capabilities cryogenic electron tomography (cryoET) microscopy (cryoEM), we determined wild-type virion. Tomograms virions...

ABSTRACT Arenaviruses exist globally and can cause hemorrhagic fever neurological diseases, exemplified by the zoonotic pathogen lymphocytic choriomeningitis virus (LCMV). The structures of individual LCMV proteins or their fragments have been reported, but architectural organization nucleocapsid assembly mechanism remain elusive. Importantly, in situ structure arenavirus fusion protein complex (glycoprotein complex, GPC) as present on virion prior to fusion, particularly with its integral...

Archaea share genomic similarities with Eukarya and cellular architectural Bacteria, though archaeal bacterial surface layers (S-layers) differ. Using cryo–electron tomography, we visualized the S-layer lattice surrounding Methanospirillum hungatei , a methanogenic archaeon. Though more compact than known structures, M. ’s is flexible hexagonal of dome-shaped tiles, uniformly spaced from both overlying cell sheath underlying membrane. Subtomogram averaging resolved hexamer tile at...

ABSTRACT Nuclear egress is an essential process in herpesviral replication whereby nascent capsids translocate from the nucleus to cytoplasm. This initial step of nuclear – budding at inner membrane coordinated by complex (NEC). Composed viral proteins UL31 and UL34, NEC deforms around capsid as latter buds into perinuclear space. oligomerization a hexagonal membrane-bound lattice for because mutations designed perturb interfaces reduce its ability. Previously, we identified suppressor...

ABSTRACT Motility of pathogenic protozoa depends on flagella (synonymous with cilia) axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules. Microtubule inner proteins (MIPs) within DMTs influence axoneme stability motility provide lineage-specific adaptations, but individual MIP functions assembly mechanisms are mostly unknown. Here, we show in the sleeping sickness parasite Trypanosoma brucei , that FAP106, a conserved at DMT junction, is required for trypanosome...