Blik is an extensible 3D visualisation tool for the annotation and analysis of cryo-electron tomography data.

Powerful, workflow-agnostic and interactive visualisation is essential for the ad hoc, human-in-the-loop workflows typical of cryo-electron tomography (cryo-ET). While several tools exist for visualisation and annotation of cryo-ET data, they are often integrated as part of monolithic processing pipelines, or focused on a specific task and offering limited reusability and extensibility. With each software suite presenting its own pros and cons and tools tailored to address specific challenges, seamless integration between available pipelines is often a difficult task. As part of the effort to enable such flexibility and move the software ecosystem towards a more collaborative and modular approach, we developed blik, an open-source napari plugin for visualisation and annotation of cryo-ET data (source code: https://github.com/brisvag/blik). blik offers fast, interactive, and user-friendly 3D visualisation thanks to napari, and is built with extensibility and modularity at the core. Data is handled and exposed through well-established scientific Python libraries such as numpy arrays and pandas dataframes. Reusable components (such as data structures, file read/write, and annotation tools) are developed as independent Python libraries to encourage reuse and community contribution. By easily integrating with established image analysis tools-even outside of the cryo-ET world-blik provides a versatile platform for interacting with cryo-ET data. On top of core visualisation features-interactive and simultaneous visualisation of tomograms, particle picks, and segmentations-blik provides an interface for interactive tools such as manual, surface-based and filament-based particle picking, and image segmentation, as well as simple filtering tools. Additional self-contained napari plugins developed as part of this work also implement interactive plotting and selection based on particle features, and label interpolation for easier segmentation. Finally, we highlight the differences with existing software and showcase blik's applicability in biological research.

The assembly of the Mitochondrial Complex I Assembly complex uncovers a redox pathway coordination.

The Mitochondrial Complex I Assembly (MCIA) complex is essential for the biogenesis of respiratory Complex I (CI), the first enzyme in the respiratory chain, which has been linked to Alzheimer's disease (AD) pathogenesis. However, how MCIA facilitates CI assembly, and how it is linked with AD pathogenesis, is poorly understood. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, releasing the FAD cofactor and converting ACAD9 from a fatty acid ß-oxidation (FAO) enzyme to a CI assembly factor. We provide evidence that ECSIT phosphorylation downregulates its association with ACAD9 and is reduced in neuronal cells upon exposure to amyloid-ß (Aß) oligomers. These findings advance our understanding of the MCIA complex assembly and suggest a possible role for ECSIT in the reprogramming of bioenergetic pathways linked to Aß toxicity, a hallmark of AD.

Structural landscape of the respiratory syncytial virus nucleocapsids.

Human Respiratory Syncytial Virus (HRSV) is a prevalent cause of severe respiratory infections in children and the elderly. The helical HRSV nucleocapsid is a template for the viral RNA synthesis and a scaffold for the virion assembly. This cryo-electron microscopy analysis reveals the non-canonical arrangement of the HRSV nucleocapsid helix, composed of 16 nucleoproteins per asymmetric unit, and the resulting systematic variations in the RNA accessibility. We demonstrate that this unique helical symmetry originates from longitudinal interactions by the C-terminal arm of the HRSV nucleoprotein. We explore the polymorphism of the nucleocapsid-like assemblies, report five structures of the full-length particles and two alternative arrangements formed by a C-terminally truncated nucleoprotein mutant, and demonstrate the functional importance of the identified longitudinal interfaces. We put all these findings in the context of the HRSV RNA synthesis machinery and delineate the structural basis for its further investigation.

Targeting structural flexibility in low density lipoprotein by integrating cryo-electron microscopy and high-speed atomic force microscopy.

Low-density lipoprotein (LDL) plays a crucial role in cholesterol metabolism. Responsible for cholesterol transport from the liver to the organs, LDL accumulation in the arteries is a primary cause of cardiovascular diseases, such as atherosclerosis. This work focuses on the fundamental question of the LDL molecular structure, as well as the topology and molecular motions of apolipoprotein B-100 (apo B-100), which is addressed by single-particle cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM). Our results suggest a revised model of the LDL core organization with respect to the cholesterol ester (CE) arrangement. In addition, a high-density region close to the flattened poles could be identified, likely enriched in free cholesterol. The most remarkable new details are two protrusions on the LDL surface, attributed to the protein apo B-100. HS-AFM adds the dimension of time and reveals for the first time a highly dynamic direct description of LDL, where we could follow large domain fluctuations of the protrusions in real time. To tackle the inherent flexibility and heterogeneity of LDL, the cryo-EM maps are further assessed by 3D variability analysis. Our study gives a detailed explanation how to approach the intrinsic flexibility of a complex system comprising lipids and protein.

EGOC inhibits TOROID polymerization by structurally activating TORC1.

Target of rapamycin complex 1 (TORC1) is a protein kinase controlling cell homeostasis and growth in response to nutrients and stresses. In Saccharomyces cerevisiae, glucose depletion triggers a redistribution of TORC1 from a dispersed localization over the vacuole surface into a large, inactive condensate called TOROID (TORC1 organized in inhibited domains). However, the mechanisms governing this transition have been unclear. Here, we show that acute depletion and repletion of EGO complex (EGOC) activity is sufficient to control TOROID distribution, independently of other nutrient-signaling pathways. The 3.9-Å-resolution structure of TORC1 from TOROID cryo-EM data together with interrogation of key interactions in vivo provide structural insights into TORC1-TORC1' and TORC1-EGOC interaction interfaces. These data support a model in which glucose-dependent activation of EGOC triggers binding to TORC1 at an interface required for TOROID assembly, preventing TORC1 polymerization and promoting release of active TORC1.

The AAA+ ATPase RavA and its binding partner ViaA modulate E. coli aminoglycoside sensitivity through interaction with the inner membrane.

Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to aminoglycosides under anaerobiosis and engaged in cell membrane regulation.

Importance of RNA length for in vitro encapsidation by the nucleoprotein of human respiratory syncytial virus.

Respiratory syncytial virus has a negative-sense single-stranded RNA genome constitutively encapsidated by the viral nucleoprotein N, forming a helical nucleocapsid which is the template for viral transcription and replication by the viral polymerase L. Recruitment of L onto the nucleocapsid depends on the viral phosphoprotein P, which is an essential L cofactor. A prerequisite for genome and antigenome encapsidation is the presence of the monomeric, RNA-free, neosynthesized N protein, named N0. Stabilization of N0 depends on the binding of the N-terminal residues of P to its surface, which prevents N oligomerization. However, the mechanism involved in the transition from N0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, the specific role of N oligomerization and RNA in the morphogenesis of viral factories, where viral transcription and replication occur, have not been elucidated although the interaction between P and N complexed to RNA has been shown to be responsible for this process. Here, using a chimeric protein comprising N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed that the nature of the 5' end of RNA does not explain the specificity of encapsidation. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories. Together, our findings provide insight into respiratory syncytial virus viral genome encapsidation specificity.

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals.

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

Structural and biochemical characterisation of the Providencia stuartii arginine decarboxylase shows distinct polymerisation and regulation.

Bacterial homologous lysine and arginine decarboxylases play major roles in the acid stress response, physiology, antibiotic resistance and virulence. The Escherichia coli enzymes are considered as their archetypes. Whereas acid stress triggers polymerisation of the E. coli lysine decarboxylase LdcI, such behaviour has not been observed for the arginine decarboxylase Adc. Here we show that the Adc from a multidrug-resistant human pathogen Providencia stuartii massively polymerises into filaments whose cryo-EM structure reveals pronounced differences between Adc and LdcI assembly mechanisms. While the structural determinants of Adc polymerisation are conserved only in certain Providencia and Burkholderia species, acid stress-induced polymerisation of LdcI appears general for enterobacteria. Analysis of the expression, activity and oligomerisation of the P. stuartii Adc further highlights the distinct properties of this unusual protein and lays a platform for future investigation of the role of supramolecular assembly in the superfamily or arginine and lysine decarboxylases.

Characterization of the Interaction Domains between the Phosphoprotein and the Nucleoprotein of Human Metapneumovirus.

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein (N), forming an RNA-N complex (NNuc), which serves as the template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping, and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to NNuc and L, allowing the attachment of the L polymerase to the NNuc template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between HMPV P and NNuc, which has not been demonstrated yet. Here, we finely characterized the binding P-NNuc interaction domains by using recombinant proteins, combined with a functional assay for the polymerase complex activity, and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to NNuc and that P binds to the N-terminal domain of N (NNTD), and we identified conserved N residues critical for the interaction. Our results allowed us to propose a structural model for the HMPV P-NNuc interaction. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Currently, no vaccine or efficient antiviral treatments are available for this pneumovirus. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work, we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction and identified a pocket on the surface of the N protein, a potential target of choice for the design of compounds interfering with N-P complexes and inhibiting viral replication.

Interactions between the Nucleoprotein and the Phosphoprotein of Pneumoviruses: Structural Insight for Rational Design of Antivirals.

Pneumoviruses include pathogenic human and animal viruses, the most known and studied being the human respiratory syncytial virus (hRSV) and the metapneumovirus (hMPV), which are the major cause of severe acute respiratory tract illness in young children worldwide, and main pathogens infecting elderly and immune-compromised people. The transcription and replication of these viruses take place in specific cytoplasmic inclusions called inclusion bodies (IBs). These activities depend on viral polymerase L, associated with its cofactor phosphoprotein P, for the recognition of the viral RNA genome encapsidated by the nucleoprotein N, forming the nucleocapsid (NC). The polymerase activities rely on diverse transient protein-protein interactions orchestrated by P playing the hub role. Among these interactions, P interacts with the NC to recruit L to the genome. The P protein also plays the role of chaperone to maintain the neosynthesized N monomeric and RNA-free (called N0) before specific encapsidation of the viral genome and antigenome. This review aims at giving an overview of recent structural information obtained for hRSV and hMPV P, N, and more specifically for P-NC and N0-P complexes that pave the way for the rational design of new antivirals against those viruses.

A flexible framework for multi-particle refinement in cryo-electron tomography.

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are increasingly used for macromolecular structure determination in situ. Here, we introduce a set of computational tools and resources designed to enable flexible approaches to STA through increased automation and simplified metadata handling. We create a bidirectional interface between the Dynamo software package and the Warp-Relion-M pipeline, providing a framework for ab initio and geometrical approaches to multiparticle refinement in M. We illustrate the power of working within this framework by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles (VLPs), and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we provide a comprehensive, step-by-step guide to obtaining a 3.4-Å reconstruction from EMPIAR-10164. The guide is hosted on https://teamtomo.org/, a collaborative online platform we establish for sharing knowledge about cryo-ET.

Proteolysis and multimerization regulate signaling along the two-component regulatory system AdeRS.

Bacterial two-component regulatory systems are ubiquitous environment-sensing signal transducers involved in pathogenesis and antibiotic resistance. The Acinetobacter baumannii two-component regulatory system AdeRS is made up of a sensor histidine kinase AdeS and a cognate response regulator AdeR, which together reduce repression of the multidrug-resistant efflux pump AdeABC. Herein we demonstrate that an N-terminal intrinsically disordered tail in AdeR is important for the upregulation of adeABC expression, although it greatly increases the susceptibility of AdeR to proteasome-mediated degradation. We also show that AdeS assembles into a hexameric state that is necessary for its full histidine kinase activity, which appears to occur via cis autophosphorylation. Taken together, this study demonstrates new structural mechanisms through which two-component systems can transduce environmental signals to impact gene expression and enlightens new potential antimicrobial approach by targeting two-component regulatory systems.

Alternative Architecture of the E. coli Chemosensory Array.

Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here, we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays appear to exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.

Structural and functional analysis of the Francisella lysine decarboxylase as a key actor in oxidative stress resistance.

Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.

Assembly of The Mitochondrial Complex†I Assembly Complex Suggests a Regulatory Role for Deflavination.

Fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are mitochondrial redox processes that generate ATP. The biogenesis of the respiratory Complex I, a 1 MDa multiprotein complex that is responsible for initiating OXPHOS, is mediated by assembly factors including the mitochondrial complex I assembly (MCIA) complex. However, the organisation and the role of the MCIA complex are still unclear. Here we show that ECSIT functions as the bridging node of the MCIA core complex. Furthermore, cryo-electron microscopy together with biochemical and biophysical experiments reveal that the C-terminal domain of ECSIT directly binds to the vestigial dehydrogenase domain of the FAO enzyme ACAD9 and induces its deflavination, switching ACAD9 from its role in FAO to an MCIA factor. These findings provide the structural basis for the MCIA complex architecture and suggest a unique molecular mechanism for coordinating the regulation of the FAO and OXPHOS pathways to ensure an efficient energy production.

Supramolecular assembly of the Escherichia coli LdcI upon acid stress.

Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of Escherichia coli LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed E. coli cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.

AAA+ ATPases: structural insertions under the magnifying glass.

AAA+ ATPases are a diverse protein superfamily which power a vast number of cellular processes, from protein degradation to genome replication and ribosome biogenesis. The latest advances in cryo-EM have resulted in a spectacular increase in the number and quality of AAA+ ATPase structures. This abundance of new information enables closer examination of different types of structural insertions into the conserved core, revealing discrepancies in the current classification of AAA+ modules into clades. Additionally, combined with biochemical data, it has allowed rapid progress in our understanding of structure-functional relationships and provided arguments both in favour and against the existence of a unifying molecular mechanism for the ATPase activity and action on substrates, stimulating further intensive research.

A paramyxovirus-like model for Ebola virus bipartite promoters.

Paramyxo- and filovirus nucleocapsids (NCs) have bipartite promoters at their 3' ends to initiate RNA synthesis. The 2 elements, promoter element 1 (PE1) and promoter element 2 (PE2), are separated by a spacer region that must be exactly a multiple of 6 nucleotides (nt) long. Paramyxovirus NCs have 13 nucleoprotein (NP) subunits/turn, such that PE1 and PE2 are juxtaposed on the same face of the NC helix, for concerted recognition by the viral polymerase. Ebola virus (EBOV) NCs, in contrast, have 25 to 28 subunits/turn, meaning that PE1 and PE2 cannot be juxtaposed. However, there is evidence that the number of subunits/turn at the 3' end of the EBOV NC is variable. We propose a paramyxovirus-like model for EBOV explaining why there are 8 contiguous copies of the PE2 repeat when 3 are sufficient, why expanding this run to 13 further improves minigenome performance, and why there is a limit to the number of hexa-nt that can be inserted in the spacer region.