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Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinsons disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

Down syndrome (DS) is a common genetic condition caused by trisomy of chromosome 21. Among their complex clinical features, including musculoskeletal, neurological, and cardiovascular disabilities, individuals with DS have an increased risk of developing progressive dementia and early-onset Alzheimer's disease (AD). This dementia is attributed to the increased gene dosage of the amyloid-β (Aβ) precursor protein gene, the formation of self-propagating Aβ and tau prion conformers, and the deposition of neurotoxic Aβ plaques and tau neurofibrillary tangles. Tau amyloid fibrils have previously been established to adopt many distinct conformations across different neurodegenerative conditions. Here, we report the characterization of brain samples from four DS cases spanning 36-63 years of age by spectral confocal imaging with conformation-specific dyes and cryo-electron microscopy (cryo-EM) to determine structures of isolated tau fibrils. High-resolution structures revealed paired helical filament (PHF) and straight filament (SF) conformations of tau that were identical to those determined from AD cases. The PHFs and SFs are made of two C-shaped protofilaments, each containing a cross-β/β-helix motif. Similar to filaments from AD cases, most filaments from the DS cases adopted the PHF form, while a minority (approximately 20%) formed SFs. Samples from the youngest individual with no documented dementia had sparse tau deposits. To isolate tau for cryo-EM from this challenging sample we used a novel affinity-grid method involving a graphene oxide surface derivatized with anti-tau antibodies. This method improved isolation and revealed that primarily tau PHFs and a minor population of chronic traumatic encephalopathy type II-like filaments were present in this youngest case. These findings expand the similarities between AD and DS to the molecular level, providing insight into their related pathologies and the potential for targeting common tau filament folds by small-molecule therapeutics and diagnostics.

In cells, microtubules (MTs) assemble from α/β‐tubulin subunits at nucleation sites containing the γ‐tubulin ring complex (γ‐TuRC). Within the γ‐TuRC, exposed γ‐tubulin molecules act as templates for MT assembly by interacting with α/β‐tubulin. The vertebrate γ‐TuRC is scaffolded by γ‐tubulin‐interacting proteins GCP2‐6 arranged in a specific order. Interestingly, the γ‐tubulin molecules in the γ‐TuRC deviate from the cylindrical geometry of MTs, raising the question of how the γ‐TuRC structure changes during MT nucleation. Recent studies on the structure of the vertebrate γ‐TuRC attached to the end of MTs came to varying conclusions. In vitro assembly of MTs, facilitated by an α‐tubulin mutant, resulted in a closed, cylindrical γ‐TuRC showing canonical interactions between all γ‐tubulin molecules and α/β‐tubulin subunits. Conversely, native MTs formed in a frog extract were capped by a partially closed γ‐TuRC, with some γ‐tubulin molecules failing to align with α/β‐tubulin. This review discusses these outcomes, along with the broader implications. [ABSTRACT FROM AUTHOR]

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds. [ABSTRACT FROM AUTHOR]

Here we review the functions of ribosomal proteins (RPs) in the nucleolar stages of large ribosomal subunit assembly in the yeast Saccharomyces cerevisiae. We summarize the effects of depleting RPs on pre-rRNA processing and turnover, on the assembly of other RPs, and on the entry and exit of assembly factors (AFs). These results are interpreted in light of recent near-atomic-resolution cryo-EM structures of multiple assembly intermediates. Results are discussed with respect to each neighborhood of RPs and rRNA. We identify several key mechanisms related to RP behavior. Neighborhoods of RPs can assemble in one or more than one step. Entry of RPs can be triggered by molecular switches, in which an AF is replaced by an RP binding to the same site. To drive assembly forward, rRNA structure can be stabilized by RPs, including clamping rRNA structures or forming bridges between rRNA domains. [ABSTRACT FROM AUTHOR]

The efficiency of an organic solar cell is highly dependent on the complex, interpenetrating morphology, and molecular order within the composite phases of the bulk heterojunction (BHJ) blend. Both these microstructural aspects are strongly influenced by the processing conditions and chemical design of donor/acceptor materials. To establish improved structure‐function relationships, it is vital to visualize the local microstructural order to provide specific local information about donor/acceptor interfaces and crystalline texture in BHJ blend films. The visualization of nanocrystallites, however, is difficult due to the complex semi‐crystalline structure with few characterization techniques capable of visualizing the molecular ordering of soft materials at the nanoscale. Here, it is demonstrated how cryo‐electron microscopy can be utilized to visualize local nanoscale order. This method is used to understand the distribution/orientation of crystallites in a BHJ blend. Long‐range (>300 nm) texturing of IEICO‐4F crystallites oriented in an edge‐on fashion is observed, which has not previously been observed for spin‐coated materials. This approach provides a wealth of quantitative information about the texture and size of nanocrystallites, which can be utilized to understand charge generation and transport in organic film. This study guides tailoring the material design and processing conditions for high‐performance organic optoelectronic devices. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses encompass a variety of established and emerging eukaryotic pathogens. Their genome replication is confined to specialized cytoplasmic membrane compartments known as replication organelles (ROs). These ROs derive from host membranes, transformed into distinct structures such as invaginated spherules or intricate membrane networks including single- and/or double-membrane vesicles. ROs play a vital role in orchestrating viral RNA synthesis and evading detection by innate immune sensors of the host. In recent years, groundbreaking cryo–electron microscopy studies conducted with several prototypic viruses have significantly advanced our understanding of RO structure and function. Notably, these studies unveiled the presence of crown-shaped multimeric viral protein complexes that seem to actively participate in viral RNA synthesis and regulate the release of newly synthesized RNA into the cytosol for translation and packaging. These findings have shed light on novel viral functions and fascinating macromolecular complexes that delineate promising new avenues for future research. [ABSTRACT FROM AUTHOR]

During the last ten years, developments in cryo–electron microscopy have transformed our understanding of eukaryotic ribosome assembly. As a result, the field has advanced from a list of the vast array of ribosome assembly factors toward an emerging molecular movie in which individual frames are represented by structures of stable ribosome assembly intermediates with complementary biochemical and genetic data. In this review, we discuss the mechanisms driving the assembly of yeast and human small and large ribosomal subunits. A particular emphasis is placed on the most recent findings that illustrate key concepts of ribosome assembly, such as folding of preribosomal RNA, the enforced chronology of assembly, enzyme-mediated irreversible transitions, and proofreading of preribosomal particles. [ABSTRACT FROM AUTHOR]

Dicer, a multi‐domain ribonuclease III (RNase III) protein, is crucial for gene regulation via RNA interference. It processes hairpin‐like precursors into microRNAs (miRNAs) and long double‐stranded RNAs (dsRNAs) into small interfering RNAs (siRNAs). During the "dicing" process, the miRNA or siRNA substrate is stably anchored and cleaved by Dicer's RNase III domain. Although numerous studies have investigated long dsRNA cleavage by Dicer, the specific mechanism by which human Dicer (hDICER) processes pre‐miRNA remains unelucidated. This review introduces the recently revealed hDICER structure bound to pre‐miRNA uncovered through cryo‐electron microscopy and compares it with previous reports describing Dicer. The domain‐wise movements of the helicase and dsRNA‐binding domain (dsRBD) and specific residues involved in substrate sequence recognition have been identified. During RNA substrate binding, the hDICER apical domains and dsRBD recognize the pre‐miRNA termini and cleavage site, respectively. Residue rearrangements in positively charged pockets within the apical domain influence substrate recognition and cleavage site determination. The specific interactions between dsRBD positively charged residues and nucleotide bases near the cleavage site emphasize the significance of cis‐acting elements in the hDICER processing mechanism. These findings provide valuable insights for understanding hDICER‐related diseases. [ABSTRACT FROM AUTHOR]

The regulation of dopamine (DA) removal from the synaptic cleft is a crucial process in neurotransmission and is facilitated by the sodium‐ and chloride‐coupled dopamine transporter DAT. Psychostimulant drugs, cocaine, and amphetamine, both block the uptake of DA, while amphetamine also triggers the release of DA. As a result, they prolong or even amplify neurotransmitter signaling. Atypical inhibitors of DAT lack cocaine‐like rewarding effects and offer a promising strategy for the treatment of drug use disorders. Here, we present the 3.2 Å resolution cryo‐electron microscopy structure of the Drosophila melanogaster dopamine transporter (dDAT) in complex with the atypical non‐competitive inhibitor AC‐4‐248. The inhibitor partially binds at the central binding site, extending into the extracellular vestibule, and locks the transporter in an outward open conformation. Our findings propose mechanisms for the non‐competitive inhibition of DAT and attenuation of cocaine potency by AC‐4‐248 and provide a basis for the rational design of more efficacious atypical inhibitors. [ABSTRACT FROM AUTHOR]

Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin. [ABSTRACT FROM AUTHOR]

We previously presented a bioinformatic method for identifying diseases that arise from a mutation in a protein's low-complexity domain that drives the protein into pathogenic amyloid fibrils. One protein so identified was the tropomyosin-receptor kinase-fused gene protein (TRK-fused gene protein or TFG). Mutations in TFG are associated with degenerative neurological conditions. Here, we present experimental evidence that confirms our prediction that these conditions are amyloid-related. We find that the low-complexity domain of TFG containing the disease-related mutations G269V or P285L forms amyloid fibrils, and we determine their structures using cryo-electron microscopy (cryo-EM). These structures are unmistakably amyloid in nature and confirm the propensity of the mutant TFG low-complexity domain to form amyloid fibrils. Also, despite resulting from a pathogenic mutation, the fibril structures bear some similarities to other amyloid structures that are thought to be nonpathogenic and even functional, but there are other factors that support these structures' relevance to disease, including an increased propensity to form amyloid compared with the wild-type sequence, structure-stabilizing influence from the mutant residues themselves, and double-protofilament amyloid cores. Our findings elucidate two potentially disease-relevant structures of a previously unknown amyloid and also show how the structural features of pathogenic amyloid fibrils may not conform to the features commonly associated with pathogenicity.

Abstract Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin.

Advances in computational tools for atomic model building are leading to accurate models of large molecular assemblies seen in electron microscopy, often at challenging resolutions of 3-4 Å. We describe new methods in the UCSF ChimeraX molecular modeling package that take advantage of machine-learning structure predictions, provide likelihood-based fitting in maps, and compute per-residue scores to identify modeling errors. Additional model-building tools assist analysis of mutations, post-translational modifications, and interactions with ligands. We present the latest ChimeraX model-building capabilities, including several community-developed extensions. ChimeraX is available free of charge for noncommercial use at https://www.rbvi.ucsf.edu/chimerax. This article is protected by copyright. All rights reserved.

Viral entry and fertilization are distinct biological processes that share a common mechanism: membrane fusion. In viral entry, enveloped viruses attach to the host cell membrane, triggering a series of conformational changes in the viral fusion proteins. This results in the exposure of a hydrophobic fusion peptide, which inserts into the host membrane and brings the viral and host membranes into close proximity. Subsequent structural rearrangements in opposing membranes lead to their fusion. Similarly, membrane fusion occurs when gametes merge during the fertilization process, though the exact mechanism remains unclear. Structural biology has played a pivotal role in elucidating the molecular mechanisms underlying membrane fusion. High-resolution structures of the viral and fertilization fusion-related proteins have provided valuable insights into the conformational changes that occur during this process. Understanding these mechanisms at a molecular level is essential for the development of antiviral therapeutics and tools to influence fertility. In this review, we will highlight the biological importance of membrane fusion and how protein structures have helped visualize both common elements and subtle divergences in the mechanisms behind fusion; in addition, we will examine the new tools that recent advances in structural biology provide researchers interested in a frame-by-frame understanding of membrane fusion.

Abstract Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo‐electron microscopy, we determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6‐diazo‐5‐oxo‐ l‐norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament‐forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent‐accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.

Abstract Down syndrome (DS) is a common genetic condition caused by trisomy of chromosome 21. Among their complex clinical features, including musculoskeletal, neurological, and cardiovascular disabilities, individuals with DS have an increased risk of developing progressive dementia and early-onset Alzheimer’s disease (AD). This dementia is attributed to the increased gene dosage of the amyloid-β (Aβ) precursor protein gene, the formation of self-propagating Aβ and tau prion conformers, and the deposition of neurotoxic Aβ plaques and tau neurofibrillary tangles. Tau amyloid fibrils have previously been established to adopt many distinct conformations across different neurodegenerative conditions. Here, we report the characterization of brain samples from four DS cases spanning 36–63 years of age by spectral confocal imaging with conformation-specific dyes and cryo–electron microscopy (cryo-EM) to determine structures of isolated tau fibrils. High-resolution structures revealed paired helical filament (PHF) and straight filament (SF) conformations of tau that were identical to those determined from AD cases. The PHFs and SFs are made of two C-shaped protofilaments, each containing a cross-β/β-helix motif. Similar to filaments from AD cases, most filaments from the DS cases adopted the PHF form, while a minority (approximately 20%) formed SFs. Samples from the youngest individual with no documented dementia had sparse tau deposits. To isolate tau for cryo-EM from this challenging sample we used a novel affinity-grid method involving a graphene oxide surface derivatized with anti-tau antibodies. This method improved isolation and revealed that primarily tau PHFs and a minor population of chronic traumatic encephalopathy type II–like filaments were present in this youngest case. These findings expand the similarities between AD and DS to the molecular level, providing insight into their related pathologies and the potential for targeting common tau filament folds by small-molecule therapeutics and diagnostics.

Objective: The 26S proteasome is a unique multicatalytic proteinase complex, which, together with the ubiquitination system, provides controlled degradation of most intracellular eukaryotic proteins. The main obstacle of studying the proteasome is its multiple intracellular forms formed due to the modularity of the proteasome assembly process and its different catalytic phenotypes. Methods: In the present study we report the first cryo-electron microscopy (cryo-EM) structure of the 26S human immunoproteasome in comparison with its constitutive form at 3.6 Å resolution. Results and Discussion: Detailed analysis of the structural features of the two complexes revealed an unveiled entrance in the outer heptameric 20S a-ring of the 26S immunoproteasome in comparison with constitutive 26S, arose due to the disjunction of the N-terminal regions of the PSMA4 and PSMA5 subunits and emerged π–π stacking interaction between the Tyr5 and Phe9 amino acid residues of the PSMA5 and PSMA6 subunits, respectively. Conclusions: We suggest that observed abolition of steric hindrance in the central channel of the 20S particle may indicate the preactivation phenotype of the human 26S immunoproteasome, even in the absence of a bound substrate. [ABSTRACT FROM AUTHOR]

α-Latrotoxin (α-LTX) was found to form two-dimensional (2D) monolayer arrays in solution at relatively low concentrations (0.1 mg/mL), with the toxin tetramer constituting a unit cell. The crystals were imaged using cryogenic electron microscopy (cryoEM), and image analysis yielded a ~12 Å projection map. At this resolution, no major conformational changes between the crystalline and solution states of α-LTX tetramers were observed. Electrophysiological studies showed that, under the conditions of crystallization, α-LTX simultaneously formed multiple channels in biological membranes that displayed coordinated gating. Two types of channels with conductance levels of 120 and 208 pS were identified. Furthermore, we observed two distinct tetramer conformations of tetramers both when observed as monodisperse single particles and within the 2D crystals, with pore diameters of 11 and 13.5 Å, suggestive of a flickering pore in the middle of the tetramer, which may correspond to the two states of toxin channels with different conductance levels. We discuss the structural changes that occur in α-LTX tetramers in solution and propose a mechanism of α-LTX insertion into the membrane. The propensity of α-LTX tetramers to form 2D crystals may explain many features of α-LTX toxicology and suggest that other pore-forming toxins may also form arrays of channels to exert maximal toxic effect. [ABSTRACT FROM AUTHOR]

The rapid emergence of multi-drug-resistant bacteria has raised a serious public health concern. Therefore, new antibiotic developments have been highly desired. Here, we propose a new method to visualize antibiotic actions on translating ribosomes in the cell-free system under macromolecular crowding conditions by cryo-electron microscopy, designated as the DARC method: the Direct visualization of Antibiotic binding on Ribosomes in the Cell-free translation system. This new method allows for acquiring a more comprehensive understanding of the mode of action of antibiotics on the translation inhibition without ribosome purification. Furthermore, with the direct link to biochemical analysis at the same condition as cryo-EM observation, we revealed the evolution of 2-DOS aminoglycosides from dibekacin (DBK) to arbekacin (ABK) by acquiring the synthetic tailored anchoring motif to lead to stronger binding affinity to ribosomes. Our cryo-EM structures of DBK and ABK bound ribosomes in the cell-free environment clearly depicted a synthetic tailored γ-amino-α-hydroxybutyryl (HABA) motif formed additional interactions with the ribosome enhancing antibiotic bindings. This new approach would be valuable for understanding the function of antibiotics for more efficient drug development. [ABSTRACT FROM AUTHOR]

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. [ABSTRACT FROM AUTHOR]

Elucidating protein conformational changes is essential because conformational changes are closely related to the functions of proteins. Cryo‐electron microscopy (cryo‐EM) experiment can be used to reconstruct protein conformational changes via a method that involves using the experimental data (two‐dimensional protein images). In this study, a reconstruction method, referred to as the "four‐dimensional imaging," was proposed. In our four‐dimensional imaging technique, the protein conformational change was obtained using the two‐dimensional protein images (the three‐dimensional electron density maps used in previously proposed techniques were not used). The protein conformation for each two‐dimensional protein image was obtained using our original protocol with molecular dynamics simulations. Using a manifold‐learning technique and two‐dimensional protein images, the protein conformations were arranged according to the conformational change of the protein. By arranging the protein conformations according to the arrangement of the protein images, four‐dimensional imaging is constructed. A simulation for a cryo‐EM experiment demonstrated the validity of our four‐dimensional imaging technique. [ABSTRACT FROM AUTHOR]

The TWIK-related acid-sensitive K+ channel 3 (TASK3) belongs to the two-pore domain (K2P) potassium channel family, which regulates cell excitability by mediating a constitutive "leak" potassium efflux in the nervous system. Extracellular acidification inhibits TASK3 channel, but the molecular mechanism by which channel inactivation is coupled to pH decrease remains unclear. Here, we report the cryo-electron microscopy structures of human TASK3 at neutral and acidic pH. Structural comparison revealed selectivity filter (SF) rearrangements upon acidification, characteristic of C-type inactivation, but with a unique structural basis. The extracellular mouth of the SF was prominently dilated and simultaneously blocked by a hydrophobic gate. His98 protonation shifted the conformational equilibrium between the conductive and C-type inactivated SF toward the latter by engaging a cation-p interaction with Trp78, consistent with molecular dynamics simulations and electrophysiological experiments. Our work illustrated how TASK3 is gated in response to extracellular pH change and implies how physiological stimuli might directly modulate the C-type gating of K2P channels. [ABSTRACT FROM AUTHOR]

The G‐protein‐coupled human cannabinoid receptor 1 (CB1) is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. The structures of CB1‐Gi complexes in synthetic agonist‐bound forms have been resolved to date. However, the commercial drug recognition and Gq coupling mechanisms of CB1 remain elusive. Herein, the cryo‐electron microscopy (cryo‐EM) structure of CB1‐Gq complex, in fenofibrate‐bound form, at near‐atomic resolution, is reported. The structure elucidates the delicate mechanisms of the precise fenofibrate recognition and Gq protein coupling by CB1 and will facilitate future drug discovery and design. [ABSTRACT FROM AUTHOR]

In addressing the challenges faced by laboratories and universities with limited (or no) cryo‐electron microscopy (cryo‐EM) infrastructure, the ESRF, in collaboration with the Grenoble Institute for Structural Biology (IBS), has implemented the cryo‐EM Solution‐to‐Structure (SOS) pipeline. This inclusive process, spanning grid preparation to high‐resolution data collection, covers single‐particle analysis and cryo‐electron tomography (cryo‐ET). Accessible through a rolling access route, proposals undergo scientific merit and technical feasibility evaluations. Stringent feasibility criteria demand robust evidence of sample homogeneity. Two distinct entry points are offered: users can either submit purified protein samples for comprehensive processing or initiate the pipeline with already vitrified cryo‐EM grids. The SOS pipeline integrates negative stain imaging (exclusive to protein samples) as a first quality step, followed by cryo‐EM grid preparation, grid screening and preliminary data collection for single‐particle analysis, or only the first two steps for cryo‐ET. In both cases, if the screening steps are successfully completed, high‐resolution data collection will be carried out using a Titan Krios microscope equipped with a latest‐generation direct electron counting detector coupled to an energy filter. The SOS pipeline thus emerges as a comprehensive and efficient solution, further democratizing access to cryo‐EM research. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) play a key role in the transduction of extracellular signals to cells and regulation of many biological processes, which makes these membrane proteins one of the most important targets for pharmacological agents. A significant increase in the number of resolved atomic structures of GPCRs has opened the possibility of developing pharmaceuticals targeting these receptors via structure-based drug design (SBDD). SBDD employs information on the structure of receptor–ligand complexes to search for selective ligands without the need for an extensive high-throughput experimental ligand screening and can significantly expand the chemical space for ligand search. In this review, we describe the process of deciphering GPCR structures using X-ray diffraction analysis and cryoelectron microscopy as an important stage in the rational design of drugs targeting this receptor class. Our main goal was to present modern developments and key features of experimental methods used in SBDD of GPCR-targeting agents to a wide range of specialists. [ABSTRACT FROM AUTHOR]

High‐resolution structures of biomolecules can be obtained using single‐particle cryo‐electron microscopy (SPA cryo‐EM), and the rapidly growing number of structures solved by this method is encouraging more researchers to utilize this technique. As with other structural biology methods, sample preparation for an SPA cryo‐EM data collection requires some expertise and an understanding of the strengths and limitations of the technique in order to make sensible decisions in the sample‐preparation process. In this article, common strategies and pitfalls are described and practical advice is given to increase the chances of success when starting an SPA cryo‐EM project. [ABSTRACT FROM AUTHOR]

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

ABSTRACTTick-borne encephalitis virus (TBEV) causes a severe disease, tick-borne encephalitis (TBE), that has a substantial epidemiological importance for Northern Eurasia. Between 10,000 and 15,000 TBE cases are registered annually despite the availability of effective formaldehyde-inactivated full-virion vaccines due to insufficient vaccination coverage, as well as sporadic cases of vaccine breakthrough. The development of improved vaccines would benefit from the atomic resolution structure of the antigen. Here we report the refined single-particle cryo-electron microscopy (cryo-EM) structure of the inactivated mature TBEV vaccine strain Sofjin–Chumakov (Far-Eastern subtype) at a resolution of 3.0 Å. The increase of the resolution with respect to the previously published structures of TBEV strains Hypr and Kuutsalo-14 (European subtype) was reached due to improvement of the virus sample quality achieved by the optimized preparation methods. All the surface epitopes of TBEV were structurally conserved in the inactivated virions. ELISA studies with monoclonal antibodies supported the hypothesis of TBEV protein shell cross-linking upon inactivation with formaldehyde.

In recent years, protein structure analysis using cryo-electron microscopy(cryo-EM) has expanded and improved. In this review, we discuss many recent improvements to the field, the problems those improvements hope to solve, and some of the still unanswered questions. Most notably, this review will discuss improvements in resolving small or fragmented protein structures, as well as methods to improve the signal-to-noise ratio of the data by increasing image contrast using carbon-based systems. We will also describe how, in the last 5 years, methodological improvements have allowed for better 3D image resolution by capturing a continuum of 3D images. We will provide examples of these methods in practice and discuss how these improved methods may be used in small-molecule drug discovery and development. © 2022 Wiley Periodicals LLC.

Francisella tularensis, a Tier 1 select agent of bioterrorism, contains a type VI secretion system (T6SS) encoded within the Francisella pathogenicity island (FPI), which is critical for its pathogenesis. Among the 18 proteins encoded by FPI is IglD, which is essential to Francisella's intracellular growth and virulence, but neither its location within T6SS nor its functional role has been established. Here, we present the cryoEM structure of IglD from Francisella novicida and show that the Francisella IglD forms a homotrimer that is structurally homologous to the T6SS baseplate protein TssK in Escherichia coli. Each IglD monomer consists of an N-terminal β-sandwich domain, a 4-helix bundle domain, and a flexible C-terminal domain. While the overall folds of IglD and TssK are similar, the two structures differ in three aspects: the relative orientation between their β-sandwich and the 4-helix bundle domains; two insertion loops present in TssK's β-sandwich domain; and, consequently, a lack of subunit-subunit interaction between insertion loops in the IglD trimer. Phylogenetic analysis indicates that IglD is genetically remote from the TssK orthologs in other T6SSs. While the other components of the Francisella baseplate are unknown, we conducted pulldown assays showing IglJ interacts with IglD and IglH, pointing to a model wherein IglD, IglH, and IglJ form the baseplate of the Francisella T6SS. Alanine substitution mutagenesis further established that IglD's hydrophobic pocket in the N-terminal β-sandwich domain interacts with two loops of IglJ, reminiscent of the TssK-TssG interaction. These results form a framework for understanding the hitherto unexplored Francisella T6SS baseplate. IMPORTANCE Francisella tularensis is a facultatively intracellular Gram-negative bacterium that causes the serious and potentially fatal zoonotic illness, tularemia. Because of its extraordinarily high infectivity and mortality to humans, especially when inhaled, F. tularensis is considered a potential bioterrorism agent and is classified as a Tier 1 select agent. The type VI secretion system (T6SS) encoded within the Francisella pathogenicity island (FPI) is critical to its pathogenesis, but its baseplate components are largely unknown. Here, we report the cryoEM structure of IglD from Francisella novicida and demonstrate its role as a component of the baseplate complex of the Francisella T6SS. We further show that IglD interacts with IglJ and IglH, and propose a model in which these proteins interact to form the Francisella T6SS baseplate. Elucidation of the structure and composition of the Francisella baseplate should facilitate the design of strategies to prevent and treat infections caused by F. tularensis.

Major Depressive Disorder (MDD) is a complex mental disorder that involves alterations in signal transmission across multiple scales and structural abnormalities. The development of effective antidepressants (ADs) has been hindered by the dominance of monoamine hypothesis, resulting in slow progress. Traditional ADs have undesirable traits like delayed onset of action, limited efficacy, and severe side effects. Recently, two categories of fast-acting antidepressant compounds have surfaced, dissociative anesthetics S-ketamine and its metabolites, as well as psychedelics such as lysergic acid diethylamide (LSD). This has led to structural research and drug development of the receptors that they target. This review provides breakthroughs and achievements in the structure of depression-related receptors and novel ADs based on these. Cryo-electron microscopy (cryo-EM) has enabled researchers to identify the structures of membrane receptors, including the N-methyl-D-aspartate receptor (NMDAR) and the 5-hydroxytryptamine 2A (5-HT2A) receptor. These high-resolution structures can be used for the development of novel ADs using virtual drug screening (VDS). Moreover, the unique antidepressant effects of 5-HT1A receptors in various brain regions, and the pivotal roles of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and tyrosine kinase receptor 2 (TrkB) in regulating synaptic plasticity, emphasize their potential as therapeutic targets. Using structural information, a series of highly selective ADs were designed based on the different role of receptors in MDD. These molecules have the favorable characteristics of rapid onset and low adverse drug reactions. This review offers researchers guidance and a methodological framework for the structure-based design of ADs. [ABSTRACT FROM AUTHOR]

Electron microscopy has played a pivotal role in elucidating the ultrastructure of membrane contact sites between cellular organelles. The advent of cryo-electron microscopy has ushered in the ability to determine atomic models of constituent proteins or protein complexes within sites of membrane contact through single particle analysis. Furthermore, it enables the visualization of the three-dimensional architecture of membrane contact sites, encompassing numerous copies of proteins, whether in vitro reconstituted or directly observed in situ using cryo-electron tomography. Nevertheless, there exists a scarcity of cryo-electron microscopy studies focused on the site of membrane contact and their constitutive proteins. This review provides an overview of the contributions made by cryo-electron microscopy to our understanding of membrane contact sites, outlines the associated limitations, and explores prospects in this field. [ABSTRACT FROM AUTHOR]

Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate. Synopsis: The cryo-EM structures of TRPV2 and TRPV5, members of the two TRPV subfamilies, in complex with the ruthenium red blocker, reveal the intimate relationship between channel gating and pore blocking. The ruthenium red is a pore blocker in both subfamilies of the TRPV channels. In TRPV2 activated by 2-APB, ruthenium red is tightly coordinated in the open selectivity filter and prevents channel inactivation through allosteric mechanism. In TRPV5 activated by PI(4,5)P2, the ruthenium red in the selectivity filter closes the lower gate by interacting with polar residues in the pore vestibule. The cryo-EM structures of TRPV2 and TRPV5, members of the two TRPV subfamilies, in complex with the ruthenium red blocker, reveal the intimate relationship between channel gating and pore blocking. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters, acting as cellular "pumps," facilitate solute translocation through membranes via ATP hydrolysis. Their overexpression is closely tied to multidrug resistance (MDR), a major obstacle in chemotherapy and neurological disorder treatment, hampering drug accumulation and delivery. Extensive research has delved into the intricate interplay between ABC transporter structure, function, and potential inhibition for MDR reversal. Cryo-electron microscopy has been instrumental in unveiling structural details of various MDR-causing ABC transporters, encompassing ABCB1, ABCC1, and ABCG2, as well as the recently revealed ABCC3 and ABCC4 structures. The newly obtained structural insight has deepened our understanding of substrate and drug binding, translocation mechanisms, and inhibitor interactions. Given the growing body of structural information available for human MDR transporters and their associated mechanisms, we believe it is timely to compile a comprehensive review of these transporters and compare their functional mechanisms in the context of multidrug resistance. Therefore, this review primarily focuses on the structural aspects of clinically significant human ABC transporters linked to MDR, with the aim of providing valuable insights to enhance the effectiveness of MDR reversal strategies in clinical therapies. [ABSTRACT FROM AUTHOR]

This paper presents new structural data about mitochondria using correlative light and electron microscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them comparable to observing animals in their natural environment on a safari. The paper highlights two areas of research that can only be accomplished using these methods. The study visualized location of the Aβ42 amyloid aggregates in relation to mitochondria to test a hypothesis of development of mitochondrial dysfunction in Alzheimer's disease. The results showed that the Aβ42 aggregates do not interact with mitochondria, although some of them are closely located. Therefore, the study demonstrated that mitochondrial dysfunction is not directly associated with the effects of aggregates on mitochondrial structure. Other processes should be considered as sources of mitochondrial dysfunction. Second unique area presented in this work is high-resolution visualization of the mitochondrial membranes and proteins in them. Analysis of the cryo-ET data reveals toroidal holes in the lamellar structures of cardiac mitochondrial cristae, where ATP synthases are located. The study proposes a new mechanism for sorting and clustering protein complexes in the membrane based on topology. According to this suggestion, position of the OXPHOS system proteins in the membrane is determined by its curvature. High-resolution tomography expands and complements existing ideas about the structural and functional organization of mitochondria. This makes it possible to study the previously inaccessible structural interactions of proteins with each other and with membranes in vivo. [ABSTRACT FROM AUTHOR]

Cryo-electron microscopy was developed as a powerful tool for imaging biological specimens in near-native conditions. Nowadays, advances in technology, equipment and computations make it possible to obtain structures of biomolecules with near-atomic resolution. Furthermore, cryo-electron tomography combined with continuous specimen tilting allows structural analysis of heterogeneous biological specimens. In particular, when combined with a cryo-focused ion beam scanning electron microscope, it becomes possible to directly analyse the structure of the biomolecules within cells, a process known as in situ cryo-electron tomography. This technique has the potential to visualize cytoplasmic zoning, involving liquid–liquid phase separation, caused by biomolecular networks in aqueous solutions, which has been the subject of recent debate. Here, we review advances in structural studies of biomolecules to study cytoplasmic zoning by in situ cryo-electron tomography. [ABSTRACT FROM AUTHOR]

Cryo‐electron microscopy (cryo‐EM) has witnessed radical progress in the past decade, driven by developments in hardware and software. While current software packages include processing pipelines that simplify the image‐processing workflow, they do not prioritize the in‐depth analysis of crucial metadata, limiting troubleshooting for challenging data sets. The widely used RELION software package lacks a graphical native representation of the underlying metadata. Here, two web‐based tools are introduced: relion_live.py, which offers real‐time feedback on data collection, aiding swift decision‐making during data acquisition, and relion_analyse.py, a graphical interface to represent RELION projects by plotting essential metadata including interactive data filtration and analysis. A useful script for estimating ice thickness and data quality during movie pre‐processing is also presented. These tools empower researchers to analyse data efficiently and allow informed decisions during data collection and processing. [ABSTRACT FROM AUTHOR]

Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated, and in bacteria, transcription initiation is mediated by sigma (σ) factors. σ recruits RNAP to the promoter DNA region, located upstream of the transcription start site (TSS) and facilitates open complex formation, where double-stranded DNA is opened up into a transcription bubble and template strand DNA is positioned inside RNAP for initial RNA synthesis. During initial transcription, RNAP remains bound to σ and upstream DNA, presumably with an enlarging transcription bubble. The release of RNAP from upstream DNA is required for promoter escape and processive transcription elongation. Bacteria sigma factors can be broadly separated into two classes with the majority belonging to the σ70 class, represented by the σ70 that regulates housekeeping genes. σ54 forms a class on its own and regulates stress response genes. Extensive studies on σ70 have revealed the molecular mechanisms of the σ70 dependent process while how σ54 transitions from initial transcription to elongation is currently unknown. Here, we present a series of cryo-electron microscopy structures of the RNAP-σ54 initial transcribing complexes with progressively longer RNA, which reveal structural changes that lead to promoter escape. Our data show that initially, the transcription bubble enlarges, DNA strands scrunch, reducing the interactions between σ54 and DNA strands in the transcription bubble. RNA extension and further DNA scrunching help to release RNAP from σ54 and upstream DNA, enabling the transition to elongation. [ABSTRACT FROM AUTHOR]

There is a variety of experimental and computational techniques available to explore protein dynamics, each presenting advantages and limitations. One promising experimental technique that is driving the development of computational methods is cryo‐electron microscopy (cryo‐EM). Cryo‐EM provides molecular‐level structural data and first estimates of conformational landscape from single particle analysis but cannot track real‐time protein dynamics and may contain uncertainties in atomic positions especially at highly dynamic regions. Molecular simulations offer atomic‐level insights into protein dynamics; however, their computing time requirements limit the conformational sampling accuracy, and it is often hard, to assess by full‐atomic simulations the cooperative movements of biological interest for large assemblies such as those resolved by cryo‐EM. Coarse‐grained (CG) simulations permit us to explore such systems, but at the costs of lower resolution and potentially incomplete sampling of conformational space. On the other hand, analytical methods may circumvent sampling limitations. In particular, elastic network models‐based normal mode analyses (ENM‐NMA) provide unique solutions for the complete mode spectra near equilibrium states, even for systems of megadaltons, and may thus deliver information on mechanisms of motions relevant to biological function. Yet, they lack atomic resolution as well as temporal information for non‐equilibrium systems. Given the complementary nature of these methods, the integration of molecular simulations and ENM‐NMA into hybrid methodologies has gained traction. This review presents the current state‐of‐the‐art in structure‐based computations and how they are helping us gain a deeper understanding of biological mechanisms, with emphasis on the development of hybrid methods accompanying the advances in cryo‐EM. This article is categorized under:Structure and Mechanism > Computational Biochemistry and Biophysics [ABSTRACT FROM AUTHOR]

Abstract The G‐protein‐coupled human cannabinoid receptor 1 (CB1) is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. The structures of CB1‐Gi complexes in synthetic agonist‐bound forms have been resolved to date. However, the commercial drug recognition and Gq coupling mechanisms of CB1 remain elusive. Herein, the cryo‐electron microscopy (cryo‐EM) structure of CB1‐Gq complex, in fenofibrate‐bound form, at near‐atomic resolution, is reported. The structure elucidates the delicate mechanisms of the precise fenofibrate recognition and Gq protein coupling by CB1 and will facilitate future drug discovery and design.

Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio1284-1959, in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer.

Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The underlying mechanism of the 2PG to PEP conversion has been studied in great detail in previous work, however that of the reverse reaction remains to be explored. Here we present structural snapshots of Mycobacterium tuberculosis (Mtb) enolase in apo, PEP-bound and two 2PG-bound forms as it catalyzes the conversion of PEP to 2PG. The two 2PG-bound complex structures differed in the conformation of the bound product (2PG) viz the widely reported canonical conformation and a novel binding pose, which we refer to here as the alternate conformation. Notably, we observed two major differences compared with the forward reaction: the presence of MgB is non-obligatory for the reaction and 2PG assumes an alternate conformation that is likely to facilitate its dissociation from the active site. Molecular dynamics studies and binding free energy calculations further substantiate that the alternate conformation of 2PG causes distortions in both metal ion coordination and hydrogen-bonding interactions, resulting in an increased flexibility of the active-site loops and aiding product release. Taken together, this study presents a probable mechanism involved in PEP to 2PG catalysis that is likely to be mediated by the conformational change of 2PG at the active site.

Virus particles consist of a protein coat that protects their genetic material and delivers it to the host cell for self-replication. Understanding the interplay between virus structure and function is a requirement for understanding critical processes in the infectious cycle such as entry, uncoating, genome metabolism, capsid assembly, maturation, and propagation. Together with well-established techniques in cell and molecular biology, physical virology has emerged as a rapidly developing field, providing detailed, novel information on the basic principles of virus assembly, disassembly, and dynamics. The Spanish research community contains a good number of groups that apply their knowledge on biology, physics, or chemistry to the study of viruses. Some of these groups got together in 2010 under the umbrella of the Spanish Interdisciplinary Network on Virus Biophysics (BioFiViNet). Thirteen years later, the network remains a fertile ground for interdisciplinary collaborations geared to reveal new aspects on the physical properties of virus particles, their role in regulating the infectious cycle, and their exploitation for the development of virus-based nanotechnology tools. Here, we highlight some achievements of Spanish groups in the field of physical virology.

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

7SK non-coding RNA (7SK) negatively regulates RNA polymerase II (RNA Pol II) elongation by inhibiting positive transcription elongation factor b (P-TEFb), and its ribonucleoprotein complex (RNP) is hijacked by HIV-1 for viral transcription and replication. Methylphosphate capping enzyme (MePCE) and La-related protein 7 (Larp7) constitutively associate with 7SK to form a core RNP, while P-TEFb and other proteins dynamically assemble to form different complexes. Here, we present the cryo-EM structures of 7SK core RNP formed with two 7SK conformations, circular and linear, and uncover a common RNA-dependent MePCE-Larp7 complex. Together with NMR, biochemical, and cellular data, these structures reveal the mechanism of MePCE catalytic inactivation in the core RNP, unexpected interactions between Larp7 and RNA that facilitate a role as an RNP chaperone, and that MePCE-7SK-Larp7 core RNP serves as a scaffold for switching between different 7SK conformations essential for RNP assembly and regulation of P-TEFb sequestration and release.

Nitrogenases have the remarkable ability to catalyze the reduction of dinitrogen to ammonia under physiological conditions. How does this happen? The current view of the nitrogenase mechanism focuses on the role of hydrides, the binding of dinitrogen in a reductive elimination process coupled to loss of dihydrogen, and the binding of substrates to a binuclear site on the active site cofactor. This review focuses on recent experimental characterizations of turnover relevant forms of the enzyme determined by cryo-electron microscopy and other approaches, and comparison of these forms to the resting state enzyme and the broader family of iron sulfur clusters. Emerging themes include the following: (i) The obligatory coupling of protein and electron transfers does not occur in synthetic and small-molecule iron–sulfur clusters. The coupling of these processes in nitrogenase suggests that they may involve unique features of the cofactor, such as hydride formation on the trigonal prismatic arrangement of irons, protonation of belt sulfurs, and/or protonation of the interstitial carbon. (ii) Both the active site cofactor and protein are dynamic under turnover conditions; the changes are such that more highly reduced forms may differ in key ways from the resting-state structure. Homocitrate appears to play a key role in coupling cofactor and protein dynamics. (iii) Structural asymmetries are observed in nitrogenase under turnover-relevant conditions by cryo-electron microscopy, although the mechanistic relevance of these states (such as half-of-sites reactivity) remains to be established. [ABSTRACT FROM AUTHOR]