Translation stalling induced mitochondrial entrapment of ribosomal quality control related proteins offers cancer cell vulnerability.

Ribosome-associated quality control (RQC) monitors ribosomes for aberrant translation. While the role of RQC in neurodegenerative disease is beginning to be appreciated, its involvement in cancer is understudied. Here, we show a positive correlation between RQC proteins ABCE1 and ZNF598 and high-grade muscle-invasive bladder cancer. Translational stalling by the inhibitor emetine (EME) leads to increased mitochondrial localization of RQC factors including ABCE1, ZNF598, and NEMF, which are continuously imported into mitochondria facilitated by increased mitochondrial membrane potential caused by EME. This reduces the availability of these factors in the cytosol, compromising the effectiveness of RQC in handling stalled ribosomes in the cytosol and those associated with the mitochondrial outer membrane (MOM). Imported RQC factors form aggregates inside the mitochondria in a process we term stalling-induced mitochondrial stress (SIMS). ABCE1 plays a crucial role in maintaining mitochondrial health during SIMS. Notably, cancer stem cells (CSCs) exhibit increased expression of ABCE1 and consequently are more resistant to EME-induced mitochondrial dysfunction. This points to a potential mechanism of drug resistance by CSCs. Our study highlights the significance of mitochondrial entrapment of RQC factors such as ABCE1 in determining the fate of cancer cells versus CSCs. Targeting ABCE1 or other RQC factors in translational inhibition cancer therapy may help overcome drug resistance.

Electrified Operando-Freezing of Electrocatalytic CO2 Reduction Cells for Cryogenic Electron Microscopy.

The ability to freeze and stabilize reaction intermediates in their metastable states and obtain their structural and chemical information with high spatial resolution is critical to advance materials technologies such as catalysis and batteries. Here, we develop an electrified operando-freezing methodology to preserve these metastable states under electrochemical reaction conditions for cryogenic electron microscopy (cryo-EM) imaging and spectroscopy. Using Cu catalysts for CO2 reduction as a model system, we observe restructuring of the Cu catalyst in a CO2 atmosphere while the same catalyst remains intact in air at the nanometer scale. Furthermore, we discover the existence of a single valence Cu (1+) state and C-O bonding at the electrified liquid-solid interface of the operando-frozen samples, which are key reaction intermediates that traditional ex situ measurements fail to detect. This work highlights our novel technique to study the local structure and chemistry of electrified liquid-solid interfaces, with broad impact beyond catalysis.

Hub stability in the calcium calmodulin-dependent protein kinase II.

The calcium calmodulin protein kinase II (CaMKII) is a multi-subunit ring assembly with a central hub formed by the association domains. There is evidence for hub polymorphism between and within CaMKII isoforms, but the link between polymorphism and subunit exchange has not been resolved. Here, we present near-atomic resolution cryogenic electron microscopy (cryo-EM) structures revealing that hubs from the α and ß isoforms, either standalone or within an ß holoenzyme, coexist as 12 and 14 subunit assemblies. Single-molecule fluorescence microscopy of Venus-tagged holoenzymes detects intermediate assemblies and progressive dimer loss due to intrinsic holoenzyme lability, and holoenzyme disassembly into dimers upon mutagenesis of a conserved inter-domain contact. Molecular dynamics (MD) simulations show the flexibility of 4-subunit precursors, extracted in-silico from the ß hub polymorphs, encompassing the curvature of both polymorphs. The MD explains how an open hub structure also obtained from the ß holoenzyme sample could be created by dimer loss and analysis of its cryo-EM dataset reveals how the gap could open further. An assembly model, considering dimer concentration dependence and strain differences between polymorphs, proposes a mechanism for intrinsic hub lability to fine-tune the stoichiometry of αß heterooligomers for their dynamic localization within synapses in neurons.

Missing Wedge Completion via Unsupervised Learning with Coordinate Networks.

Cryogenic electron tomography (cryoET) is a powerful tool in structural biology, enabling detailed 3D imaging of biological specimens at a resolution of nanometers. Despite its potential, cryoET faces challenges such as the missing wedge problem, which limits reconstruction quality due to incomplete data collection angles. Recently, supervised deep learning methods leveraging convolutional neural networks (CNNs) have considerably addressed this issue; however, their pretraining requirements render them susceptible to inaccuracies and artifacts, particularly when representative training data is scarce. To overcome these limitations, we introduce a proof-of-concept unsupervised learning approach using coordinate networks (CNs) that optimizes network weights directly against input projections. This eliminates the need for pretraining, reducing reconstruction runtime by 3 - 20× compared to supervised methods. Our in silico results show improved shape completion and reduction of missing wedge artifacts, assessed through several voxel-based image quality metrics in real space and a novel directional Fourier Shell Correlation (FSC) metric. Our study illuminates benefits and considerations of both supervised and unsupervised approaches, guiding the development of improved reconstruction strategies.

Missing Wedge Completion via Unsupervised Learning with Coordinate Networks.

Cryogenic electron tomography (cryoET) is a powerful tool in structural biology, enabling detailed 3D imaging of biological specimens at a resolution of nanometers. Despite its potential, cryoET faces challenges such as the missing wedge problem, which limits reconstruction quality due to incomplete data collection angles. Recently, supervised deep learning methods leveraging convolutional neural networks (CNNs) have considerably addressed this issue; however, their pretraining requirements render them susceptible to inaccuracies and artifacts, particularly when representative training data is scarce. To overcome these limitations, we introduce a proof-of-concept unsupervised learning approach using coordinate networks (CNs) that optimizes network weights directly against input projections. This eliminates the need for pretraining, reducing reconstruction runtime by 3-20× compared to supervised methods. Our in silico results show improved shape completion and reduction of missing wedge artifacts, assessed through several voxel-based image quality metrics in real space and a novel directional Fourier Shell Correlation (FSC) metric. Our study illuminates benefits and considerations of both supervised and unsupervised approaches, guiding the development of improved reconstruction strategies.

Dramatic Changes in Mitochondrial Subcellular Location and Morphology Accompany Activation of the CO2 Concentrating Mechanism.

Dynamic changes in intracellular ultrastructure can be critical for the ability of organisms to acclimate to environmental conditions. Microalgae, which are responsible for ~50% of global photosynthesis, compartmentalize their Rubisco into a specialized structure known as the pyrenoid when the cells experience limiting CO2 conditions; this compartmentalization appears to be a component of the CO2 Concentrating Mechanism (CCM), which facilitates photosynthetic CO2 fixation as environmental levels of inorganic carbon (Ci) decline. Changes in the spatial distribution of mitochondria in green algae have also been observed under CO2 limiting conditions, although a role for this reorganization in CCM function remains unclear. We used the green microalgae Chlamydomonas reinhardtii to monitor changes in the position and ultrastructure of mitochondrial membranes as cells transition between high CO2 (HC) and Low/Very Low CO2 (LC/VLC). Upon transferring cells to VLC, the mitochondria move from a central to a peripheral location, become wedged between the plasma membrane and chloroplast envelope, and mitochondrial membranes orient in parallel tubular arrays that extend from the cell's apex to its base. We show that these ultrastructural changes require protein and RNA synthesis, occur within 90 min of shifting cells to VLC conditions, correlate with CCM induction and are regulated by the CCM master regulator CIA5. The apico-basal orientation of the mitochondrial membrane, but not the movement of the mitochondrion to the cell periphery, is dependent on microtubules and the MIRO1 protein, which is involved in membrane-microtubule interactions. Furthermore, blocking mitochondrial electron transport in VLC acclimated cells reduces the cell's affinity for inorganic carbon. Overall, our results suggest that CIA5-dependent mitochondrial repositioning/reorientation functions in integrating cellular architecture and energetics with CCM activities and invite further exploration of how intracellular architecture can impact fitness under dynamic environmental conditions.

Cryogenic electron microscopy and tomography reveal imperfect icosahedral symmetry in alphaviruses.

Alphaviruses are spherical, enveloped RNA viruses with two-layered icosahedral architecture. The structures of many alphaviruses have been studied using cryogenic electron microscopy (cryo-EM) reconstructions, which impose icosahedral symmetry on the viral particles. Using cryogenic electron tomography (cryo-ET), we revealed a polarized symmetry defect in the icosahedral lattice of Chikungunya virus (CHIKV) in situ, similar to the late budding particles, suggesting the inherent imperfect symmetry originates from the final pinch-off of assembled virions. We further demonstrated this imperfect symmetry is also present in in vitro purified CHIKV and Mayaro virus, another arthritogenic alphavirus. We employed a subparticle-based single-particle analysis protocol to circumvent the icosahedral imperfection and boosted the resolution of the structure of the CHIKV to ∼3 Šresolution, which revealed detailed molecular interactions between glycoprotein E1-E2 heterodimers in the transmembrane region and multiple lipid-like pocket factors located in a highly conserved hydrophobic pocket. This complementary use of in situ cryo-ET and single-particle cryo-EM approaches provides a more precise structural description of near-icosahedral viruses and valuable insights to guide the development of structure-based antiviral therapies against alphaviruses.

Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum.

Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells. IMPORTANCE: Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.

Tertiary folds of the SL5 RNA from the 5' proximal region of SARS-CoV-2 and related coronaviruses.

Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2), resolved at 4.7 Å resolution, exhibits a T-shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the T's "arms." Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4 to 6.9 Å resolution, respectively) indicate that the junction geometry and inter-hexaloop distances are conserved features across these human-infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non-human-infecting betacoronavirus BtCoV-HKU5 (5.9 to 8.0 Å resolution). SL5s from human-infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4 to 9.0 Å resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU-capped arms, but with a phylogenetically distinct crossing angle, an X-shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross-genus similarities and notable differences, with implications for potential protein-binding modes and therapeutic targets.

CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism.

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct conformations involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway. This peptide is highly conserved among species variants of CLC-2 but is not present in other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a "ball-and-chain" gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

Improving resolution and resolvability of single-particle cryoEM structures using Gaussian mixture models.

Cryogenic electron microscopy is widely used in structural biology, but its resolution is often limited by the dynamics of the macromolecule. Here we developed a refinement protocol based on Gaussian mixture models that integrates particle orientation and conformation estimation and improves the alignment for flexible domains of protein structures. We demonstrated this protocol on multiple datasets, resulting in improved resolution and resolvability, locally and globally, by visual and quantitative measures.

Targeted mutagenesis of the herpesvirus fusogen central helix captures transition states.

Herpesviruses remain a burden for animal and human health, including the medically important varicella-zoster virus (VZV). Membrane fusion mediated by conserved core glycoproteins, the fusogen gB and the heterodimer gH-gL, enables herpesvirus cell entry. The ectodomain of gB orthologs has five domains and is proposed to transition from a prefusion to postfusion conformation but the functional relevance of the domains for this transition remains poorly defined. Here we describe structure-function studies of the VZV gB DIII central helix targeting residues 526EHV528. Critically, a H527P mutation captures gB in a prefusion conformation as determined by cryo-EM, a loss of membrane fusion in a virus free assay, and failure of recombinant VZV to spread in cell monolayers. Importantly, two predominant cryo-EM structures of gB[H527P] are identified by 3D classification and focused refinement, suggesting they represented gB conformations in transition. These studies reveal gB DIII as a critical element for herpesvirus gB fusion function.

Tertiary folds of the SL5 RNA from the 5' proximal region of SARS-CoV-2 and related coronaviruses.

Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically-determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus SARS-CoV-2, resolved at 4.7 Å resolution, exhibits a T-shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the T's "arms." Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4-6.9 Å resolution, respectively) indicate that the junction geometry and inter-hexaloop distances are conserved features across the studied human-infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non-human-infecting betacoronavirus BtCoV-HKU5 (5.9-8.0 Å resolution). SL5s from human-infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4-9.0 Å resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU-capped arms, but with a phylogenetically distinct crossing angle, an X-shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross-genus similarities, with implications for potential protein-binding modes and therapeutic targets.

Structural insights into the modulation of coronavirus spike tilting and infectivity by hinge glycans.

Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context.

Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy a platform for studying cardiovascular disease by combining cryo-ET with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). After developing a cryo-ET workflow for visualizing macromolecules in hiPSC-CMs, we reconstructed sub-nanometer resolution structures of the human thin filament, a central component of the contractile machinery. We also visualized a previously unobserved organization of a regulatory complex that connects muscle contraction to calcium signaling (the troponin complex), highlighting the value of our approach for interrogating the structures of cardiac proteins in their cellular context.

The next decade in structural biology.

As we celebrate the 30th anniversary of Structure, we have asked structural biologists about their expectations on how their respective fields are likely to develop in the next ten years in this collection of Voices.

Improving Resolution and Resolvability of Single Particle CryoEM using Gaussian Mixture Models.

Cryogenic electron microscopy is widely used in structural biology, but its resolution is often limited by the dynamics of the macromolecule. Here, we developed a refinement protocol based on Gaussian mixture models that integrates particle orientation and conformation estimation, and improves the alignment for flexible domains of protein structures. We demonstrated this protocol on multiple datasets, resulting in improved resolution and resolvability, locally and globally, by visual and quantitative measures.

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism.

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a "ball-and-chain" gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

RNA target highlights in CASP15: Evaluation of predicted models by structure providers.

The first RNA category of the Critical Assessment of Techniques for Structure Prediction competition was only made possible because of the scientists who provided experimental structures to challenge the predictors. In this article, these scientists offer a unique and valuable analysis of both the successes and areas for improvement in the predicted models. All 10 RNA-only targets yielded predictions topologically similar to experimentally determined structures. For one target, experimentalists were able to phase their x-ray diffraction data by molecular replacement, showing a potential application of structure predictions for RNA structural biologists. Recommended areas for improvement include: enhancing the accuracy in local interaction predictions and increased consideration of the experimental conditions such as multimerization, structure determination method, and time along folding pathways. The prediction of RNA-protein complexes remains the most significant challenge. Finally, given the intrinsic flexibility of many RNAs, we propose the consideration of ensemble models.