TomoNet: A streamlined cryogenic electron tomography software pipeline with automatic particle picking on flexible lattices.

Cryogenic electron tomography (cryoET) is capable of determining in situ biological structures of molecular complexes at near-atomic resolution by averaging half a million subtomograms. While abundant complexes/particles are often clustered in arrays, precisely locating and seamlessly averaging such particles across many tomograms present major challenges. Here, we developed TomoNet, a software package with a modern graphical user interface to carry out the entire pipeline of cryoET and subtomogram averaging to achieve high resolution. TomoNet features built-in automatic particle picking and three-dimensional (3D) classification functions and integrates commonly used packages to streamline high-resolution subtomogram averaging for structures in 1D, 2D, or 3D arrays. Automatic particle picking is accomplished in two complementary ways: one based on template matching and the other using deep learning. TomoNet's hierarchical file organization and visual display facilitate efficient data management as required for large cryoET datasets. Applications of TomoNet to three types of datasets demonstrate its capability of efficient and accurate particle picking on flexible and imperfect lattices to obtain high-resolution 3D biological structures: virus-like particles, bacterial surface layers within cellular lamellae, and membranes decorated with nuclear egress protein complexes. These results demonstrate TomoNet's potential for broad applications to various cryoET projects targeting high-resolution in situ structures.

Structure-guided mutagenesis targeting interactions between pp150 tegument protein and small capsid protein identify five lethal and two live-attenuated HCMV mutants.

Human cytomegalovirus (HCMV) replication relies on a nucleocapsid coat of the 150 kDa, subfamily-specific tegument phosphoprotein (pp150) to regulate cytoplasmic virion maturation. While recent structural studies revealed pp150-capsid interactions, the role of specific amino-acids involved in these interactions have not been established experimentally. In this study, pp150 and the small capsid protein (SCP), one of pp150's binding partners found atop the major capsid protein (MCP), were subjected to mutational and structural analyses. Mutations to clusters of polar or hydrophobic residues along the pp150-SCP interface abolished viral replication, with no replication detected in mutant virus-infected cells. Notably, a single amino acid mutation (pp150 K255E) at the pp150-MCP interface significantly attenuated viral replication, unlike in pp150-deletion mutants where capsids degraded outside host nuclei. These functionally significant mutations targeting pp150-capsid interactions, particularly the pp150 K255E replication-attenuated mutant, can be explored to overcome the historical challenges of developing effective antivirals and vaccines against HCMV infection.

Overcoming the preferred orientation problem in cryoEM with self-supervised deep-learning.

While advances in single-particle cryoEM have enabled the structural determination of macromolecular complexes at atomic resolution, particle orientation bias (the so-called "preferred" orientation problem) remains a complication for most specimens. Existing solutions have relied on biochemical and physical strategies applied to the specimen and are often complex and challenging. Here, we develop spIsoNet, an end-to-end self-supervised deep-learning-based software to address the preferred orientation problem. Using preferred-orientation views to recover molecular information in under-sampled views, spIsoNet improves both angular isotropy and particle alignment accuracy during 3D reconstruction. We demonstrate spIsoNet's capability of generating near-isotropic reconstructions from representative biological systems with limited views, including ribosomes, ß-galactosidases, and a previously intractable hemagglutinin trimer dataset. spIsoNet can also be generalized to improve map isotropy and particle alignment of preferentially oriented molecules in subtomogram averaging. Therefore, without additional specimen-preparation procedures, spIsoNet provides a general computational solution to the preferred orientation problem.

Atomic structures of a bacteriocin targeting Gram-positive bacteria.

Due to envelope differences between Gram-positive and Gram-negative bacteria1, engineering precision bactericidal contractile nanomachines2 requires atomic-level understanding of their structures; however, only those killing a Gram-negative bacterium are currently known3,4. Here, we report the atomic structures of an engineered diffocin, a contractile syringe-like molecular machine that kills the Gram-positive bacterium Clostridioides difficile. Captured in one pre-contraction and two post-contraction states, each structure fashions six proteins in the bacteria-targeting baseplate, two proteins in the energy-storing trunk, and a collar protein linking the sheath with the membrane-penetrating tube. Compared to contractile machines targeting Gram-negative bacteria, major differences reside in the baseplate and contraction magnitude, consistent with differences between their targeted envelopes. The multifunctional hub-hydrolase protein connects the tube and baseplate and is positioned to degrade peptidoglycan during penetration. The full-length tape measure protein forms a coiled-coil helix bundle homotrimer spanning the entire length of the diffocin. Our study offers mechanical insights and principles for designing potent protein-based precision antibiotics.

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.

The recognition of 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5' ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yLuc7 protein (to mimic the lack of Luc7Ls in human U1 snRNP) and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast.

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.

TomoNet: A streamlined cryoET software pipeline with automatic particle picking on flexible lattices.

Cryogenic electron tomography (cryoET) is capable of determining in situ biological structures of molecular complexes at near atomic resolution by averaging half a million subtomograms. While abundant complexes/particles are often clustered in arrays, precisely locating and seamlessly averaging such particles across many tomograms present major challenges. Here, we developed TomoNet, a software package with a modern graphical user interface to carry out the entire pipeline of cryoET and subtomogram averaging to achieve high resolution. TomoNet features built-in automatic particle picking and 3D classification functions and integrates commonly used packages to streamline high-resolution subtomogram averaging for structures in one-, two- or three-dimensional arrays. Automatic particle picking is accomplished in two complementary ways: one based on template matching and the other employing deep learning. TomoNet's hierarchical file organization and visual display facilitate efficient data management as required for large cryoET datasets. Applications of TomoNet to three types of datasets demonstrate its capability of efficient and accurate particle picking on flexible and imperfect lattices to obtain high-resolution 3D biological structures: virus-like particles, bacterial surface layers within cellular lamellae, and membranes decorated with nuclear egress protein complexes. These results demonstrate TomoNet's potential for broad applications to various cryoET projects targeting high-resolution in situ structures.

Structure-Guided Mutagenesis Targeting Interactions between pp150 Tegument Protein and Small Capsid Protein Identify Five Lethal and Two Live Attenuated HCMV Mutants.

Human cytomegalovirus (HCMV) replication relies on a nucleocapsid coat of the 150kDa, subfamily-specific tegument phosphoprotein (pp150) to regulate cytoplasmic virion maturation. While recent structural studies revealed pp150-capsid interactions, the role of specific amino-acids involved in these interactions have not been established experimentally. In this study, pp150 and the small capsid protein (SCP), one of pp150's binding partners found atop the major capsid protein (MCP), were subjected to mutational and structural analyses. Mutations to clusters of polar or hydrophobic residues along the pp150-SCP interface abolished viral replication, with no replication detected in mutant virus-infected cells. Notably, a single point mutation at the pp150-MCP interface significantly attenuated viral replication, unlike the situation of pp150-deletion mutation where capsids degraded outside host nuclei. These functionally significant mutations targeting pp150-capsid interactions, particularly the pp150 K255E replication-attenuated mutant, can be explored to overcome the historical challenges of developing effective antivirals and vaccines against HCMV infection.

The incredible bulk: Human cytomegalovirus tegument architectures uncovered by AI-empowered cryo-EM.

The compartmentalization of eukaryotic cells presents considerable challenges to the herpesvirus life cycle. The herpesvirus tegument, a bulky proteinaceous aggregate sandwiched between herpesviruses' capsid and envelope, is uniquely evolved to address these challenges, yet tegument structure and organization remain poorly characterized. We use deep-learning-enhanced cryogenic electron microscopy to investigate the tegument of human cytomegalovirus virions and noninfectious enveloped particles (NIEPs; a genome packaging-aborted state), revealing a portal-biased tegumentation scheme. We resolve atomic structures of portal vertex-associated tegument (PVAT) and identify multiple configurations of PVAT arising from layered reorganization of pUL77, pUL48 (large tegument protein), and pUL47 (inner tegument protein) assemblies. Analyses show that pUL77 seals the last-packaged viral genome end through electrostatic interactions, pUL77 and pUL48 harbor a head-linker-capsid-binding motif conducive to PVAT reconfiguration, and pUL47/48 dimers form 45-nm-long filaments extending from the portal vertex. These results provide a structural framework for understanding how herpesvirus tegument facilitates and evolves during processes spanning viral genome packaging to delivery.

The universal suppressor mutation restores membrane budding defects in the HSV-1 nuclear egress complex by stabilizing the oligomeric lattice.

Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress-budding at the inner nuclear membrane-is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.

Structural basis for polyuridine tract recognition by SARS-CoV-2 Nsp15.

SARS-CoV-2 non-structural protein 15 (Nsp15) is critical for productive viral replication and evasion of host immunity. The uridine-specific endoribonuclease activity of Nsp15 mediates the cleavage of the polyuridine [poly(U)] tract of the negative-strand coronavirus genome to minimize the formation of dsRNA that activates the host antiviral interferon signaling. However, the molecular basis for the recognition and cleavage of the poly(U) tract by Nsp15 is incompletely understood. Here, we present cryogenic electron microscopy (cryoEM) structures of SARS-CoV-2 Nsp15 bound to viral replication intermediate dsRNA containing poly(U) tract at 2.7-3.3 Å resolution. The structures reveal one copy of dsRNA binds to the sidewall of an Nsp15 homohexamer, spanning three subunits in two distinct binding states. The target uracil is dislodged from the base-pairing of the dsRNA by amino acid residues W332 and M330 of Nsp15, and the dislodged base is entrapped at the endonuclease active site center. Up to 20 A/U base pairs are anchored on the Nsp15 hexamer, which explains the basis for a substantially shortened poly(U) sequence in the negative strand coronavirus genome compared to the long poly(A) tail in its positive strand. Our results provide mechanistic insights into the unique immune evasion strategy employed by coronavirus Nsp15.

Exosomes from Von Hippel-Lindau-Null Cancer Cells Promote Metastasis in Renal Cell Carcinoma.

Exosomes are extracellular vesicles that modulate essential physiological and pathological signals. Communication between cancer cells that express the von Hippel-Lindau (VHL) tumor suppressor gene and those that do not is instrumental to distant metastasis in renal cell carcinoma (RCC). In a novel metastasis model, VHL(-) cancer cells are the metastatic driver, while VHL(+) cells receive metastatic signals from VHL(-) cells and undergo aggressive transformation. This study investigates whether exosomes could be mediating metastatic crosstalk. Exosomes isolated from paired VHL(+) and VHL(-) cancer cell lines were assessed for physical, biochemical, and biological characteristics. Compared to the VHL(+) cells, VHL(-) cells produce significantly more exosomes that augment epithelial-to-mesenchymal transition (EMT) and migration of VHL(+) cells. Using a Cre-loxP exosome reporter system, the fluorescent color conversion and migration were correlated with dose-dependent delivery of VHL(-) exosomes. VHL(-) exosomes even induced a complete cascade of distant metastasis when added to VHL(+) tumor xenografts in a duck chorioallantoic membrane (dCAM) model, while VHL(+) exosomes did not. Therefore, this study supports that exosomes from VHL(-) cells could mediate critical cell-to-cell crosstalk to promote metastasis in RCC.

Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM.

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids, but the reasons for such misbehavior are not well understood, limiting systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy - rationalizing the use of surfactant, which is a direct solution to reducing the surface tension of the aqueous solution. By conducting cryogenic electron tomography (cryoET) with the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradual reduction of the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that applicable surfactants do not damage the biological complex, thus suggesting that they might offer a practical, simple, and general solution to the problem for high-resolution cryoEM. Application of this solution to a real-world AWI adsorption problem with a more challenging membrane protein, namely, the ClC-1 channel, has led to its first near-atomic structure using cryoEM.

Hierarchical organization and assembly of the archaeal cell sheath from an amyloid-like protein.

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 organization of the prototypical archaeon, Methanospirillum hungatei. Fitting of Alphafold-predicted model of the sheath protein (SH) monomer into the 7.9 Å-resolution structure reveals that the sheath cylinder consists of axially stacked ß-hoops, each of which is comprised of two to six 400 nm-diameter rings of ß-strand arches (ß-rings). With both similarities to and differences from amyloid cross-ß fibril architecture, each ß-ring contains two giant ß-sheets contributed by ~ 450 SH monomers that entirely encircle the outer circumference of the cell. Tomograms of immature cells suggest models of sheath biogenesis: oligomerization of SH monomers into ß-ring precursors after their membrane-proximal cytoplasmic synthesis, followed by translocation through the unplugged end of a dividing cell, and insertion of nascent ß-hoops into the immature sheath cylinder at the junction of two daughter cells.

Preparation and stability of pegylated poly(S-alkyl-L-homocysteine) coacervate core micelles in aqueous media.

We report development and preparation of synthetic polypeptide based, coacervate core polyelectrolyte complex micelles, PCMs, in aqueous media, which were characterized and evaluated for the encapsulation and in vitro release of a model single-stranded RNA, polyadenylic acid, poly(A). Cationic, α-helical polypeptides pegylated at their N-termini, PEG113-b-5bn and PEG113-b-5cn, were designed to form coacervate core PCMs upon mixing with multivalent anions in aqueous media. Sodium tripolyphosphate (TPP) and poly(A) were used as model multivalent anions that allowed optimization of polypeptide composition and chain length for formation of stable, nanoscale PCMs. PEG113-b-5c27 was selected for preparation of PCMs that were characterized under different environmental conditions using dynamic light scattering, atomic force microscopy and cryoelectron microscopy. The PCMs were found to efficiently encapsulate poly(A), were stable at physiologically relevant pH and solution ionic strength, and were able to release poly(A) in the presence of excess polyvalent anions. These PCMs were found to be a promising model system for further development of polypeptide based therapeutic delivery vehicles.

FAP106 is an interaction hub for assembling microtubule inner proteins at the cilium inner junction.

Motility of pathogenic protozoa depends on flagella (synonymous with cilia) with axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules. Microtubule inner proteins (MIPs) within DMTs influence axoneme stability and motility and provide lineage-specific adaptations, but individual MIP functions and assembly mechanisms are mostly unknown. Here, we show in the sleeping sickness parasite Trypanosoma brucei, that FAP106, a conserved MIP at the DMT inner junction, is required for trypanosome motility and functions as a critical interaction hub, directing assembly of several conserved and lineage-specific MIPs. We use comparative cryogenic electron tomography (cryoET) and quantitative proteomics to identify MIP candidates. Using RNAi knockdown together with fitting of AlphaFold models into cryoET maps, we demonstrate that one of these candidates, MC8, is a trypanosome-specific MIP required for parasite motility. Our work advances understanding of MIP assembly mechanisms and identifies lineage-specific motility proteins that are attractive targets to consider for therapeutic intervention.

Structural basis of HIV-1 Vif-mediated E3 ligase targeting of host APOBEC3H.

Human APOBEC3 (A3) cytidine deaminases are antiviral factors that are particularly potent against retroviruses. As a countermeasure, HIV-1 uses a viral infectivity factor (Vif) to target specific human A3s for proteasomal degradation. Vif recruits cellular transcription cofactor CBF-ß and Cullin-5 (CUL5) RING E3 ubiquitin ligase to bind different A3s distinctively, but how this is accomplished remains unclear in the absence of the atomic structure of the complex. Here, we present the cryo-EM structures of HIV-1 Vif in complex with human A3H, CBF-ß and components of CUL5 ubiquitin ligase (CUL5, ELOB, and ELOC). Vif nucleates the entire complex by directly binding four human proteins, A3H, CBF-ß, CUL5, and ELOC. The structures reveal a large interface area between A3H and Vif, primarily mediated by an α-helical side of A3H and a five-stranded ß-sheet of Vif. This A3H-Vif interface unveils the basis for sensitivity-modulating polymorphism of both proteins, including a previously reported gain-of-function mutation in Vif isolated from HIV/AIDS patients. Our structural and functional results provide insights into the remarkable interplay between HIV and humans and would inform development efforts for anti-HIV therapeutics.

Structural basis of gRNA stabilization and mRNA recognition in trypanosomal RNA editing.

In Trypanosoma brucei, the editosome, composed of RNA-editing substrate-binding complex (RESC) and RNA-editing catalytic complex (RECC), orchestrates guide RNA (gRNA)-programmed editing to recode cryptic mitochondrial transcripts into messenger RNAs (mRNAs). The mechanism of information transfer from gRNA to mRNA is unclear owing to a lack of high-resolution structures for these complexes. With cryo-electron microscopy and functional studies, we have captured gRNA-stabilizing RESC-A and gRNA-mRNA-binding RESC-B and RESC-C particles. RESC-A sequesters gRNA termini, thus promoting hairpin formation and blocking mRNA access. The conversion of RESC-A into RESC-B or -C unfolds gRNA and allows mRNA selection. The ensuing gRNA-mRNA duplex protrudes from RESC-B, likely exposing editing sites to RECC-catalyzed cleavage, uridine insertion or deletion, and ligation. Our work reveals a remodeling event facilitating gRNA-mRNA hybridization and assembly of a macromolecular substrate for the editosome's catalytic modality.