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The disease of transthyretin (TTR) amyloidosis (ATTR) has been known since the 1960s, and during the past 60 or so years, there has been a sustained period of steady discoveries that have led to the current model of ATTR pathogenesis. More recent research has achieved major advances in both diagnostics and therapeutics for ATTR, which are having a significant impact on ATTR patients today. Aiding these recent achievements has been the remarkable ability of cryo-electron microscopy (EM) to determine high-resolution structures of amyloid fibrils obtained from individual patients. Here, we will examine the cryo-EM structures of transthyretin amyloid fibrils to explore the structural basis of the two monoclonal antibody therapies for ATTR that are in clinical trials, ALXN-2220 and Coramitug, as well as to point out potential applications of this approach to other systemic amyloid diseases. [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]

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]

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]

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]

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]

Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A "linear" 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect. [ABSTRACT FROM AUTHOR]

The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the reaction. This involves shifts in the equilibrium of the oligomeric states of the enzyme, but how these are achieved and result in changes in enzyme catalytic competence has yet to be determined. Here, the regulation of human β-ureidopropionase was further explored via site-directed mutagenesis, inhibition studies, and cryo-electron microscopy. The active-site residue E207, as well as H173 and H307 located at the dimer–dimer interface, are shown to play crucial roles in enzyme activation. Dimer association to larger assemblies requires closure of active-site loops, which positions the catalytically crucial E207 stably in the active site. H173 and H307 likely respond to ligand-induced changes in their environment with changes in their protonation states, which fine-tunes the active-site loop stability and the strength of dimer–dimer interfaces and explains the previously observed pH influence on the oligomer equilibrium. The correlation between substrate analogue structure and effect on enzyme assembly suggests that the ability to favourably interact with F205 may distinguish activators from inhibitors. The cryo-EM structure of human β-ureidopropionase assembly obtained at low pH provides first insights into the architecture of its activated state. and validates our current model of the allosteric regulation mechanism. Closed entrance loop conformations and dimer–dimer interfaces are highly conserved between human and fruit fly enzymes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aptamers are synthetic nucleic acids that are developed to target with high affinity and specificity chemical entities ranging from single ions to macromolecules and present a wide range of chemical and physical properties. Their ability to selectively bind proteins has made these compounds very attractive and versatile tools, in both basic and applied sciences, to such an extent that they are considered an appealing alternative to antibodies. Here, by exhaustively surveying the content of the Protein Data Bank (PDB), we review the structural aspects of the protein–aptamer recognition process. As a result of three decades of structural studies, we identified 144 PDB entries containing atomic-level information on protein–aptamer complexes. Interestingly, we found a remarkable increase in the number of determined structures in the last two years as a consequence of the effective application of the cryo-electron microscopy technique to these systems. In the present paper, particular attention is devoted to the articulated architectures that protein–aptamer complexes may exhibit. Moreover, the molecular mechanism of the binding process was analyzed by collecting all available information on the structural transitions that aptamers undergo, from their protein-unbound to the protein-bound state. The contribution of computational approaches in this area is also highlighted. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, membrane proteins play a crucial role. They fall into three categories: intrinsic proteins, extrinsic proteins, and proteins that are essential to the human genome (30% of which is devoted to encoding them). Hydrophobic interactions inside the membrane serve to stabilize integral proteins, which span the lipid bilayer. This review investigates a number of computational and experimental methods used to study membrane proteins. It encompasses a variety of technologies, including electrophoresis, X-ray crystallography, cryogenic electron microscopy (cryo-EM), nuclear magnetic resonance spectroscopy (NMR), biophysical methods, computational methods, and artificial intelligence. The link between structure and function of membrane proteins has been better understood thanks to these approaches, which also hold great promise for future study in the field. The significance of fusing artificial intelligence with experimental data to improve our comprehension of membrane protein biology is also covered in this paper. This effort aims to shed light on the complexity of membrane protein biology by investigating a variety of experimental and computational methods. Overall, the goal of this review is to emphasize how crucial it is to understand the functions of membrane proteins in eukaryotic cells. It gives a general review of the numerous methods used to look into these crucial elements and highlights the demand for multidisciplinary approaches to advance our understanding. [ABSTRACT FROM AUTHOR]

With the increasing popularity of cryo-electron tomography (cryo-ET) in recent years, the quest to establish a method for growing primary neurons directly on electron microscopy grids (EM grids) has been ongoing. Here we describe a straightforward way to establish a mature neuronal network on EM grids, which includes formation of synaptic contacts. These synapses were thin enough to allow for direct visualization of small filaments such as SNARE proteins tethering the synaptic vesicle (SV) to the active zone plasma membrane on a Titan Krios without prior focused ion-beam milling. [ABSTRACT FROM AUTHOR]

Striated muscle thick filaments are composed of myosin II and several non-myosin proteins which define the filament length and modify its function. Myosin II has a globular N-terminal motor domain comprising its catalytic and actin-binding activities and a long α -helical, coiled tail that forms the dense filament backbone. Myosin alone polymerizes into filaments of irregular length, but striated muscle thick filaments have defined lengths that, with thin filaments, define the sarcomere structure. The motor domain structure and function are well understood, but the myosin filament backbone is not. Here we report on the structure of the flight muscle thick filaments from Drosophila melanogaster at 4.7 Å resolution, which eliminates previous ambiguities in non-myosin densities. The full proximal S2 region is resolved, as are the connecting densities between the Ig domains of stretchin-klp. The proteins, flightin, and myofilin are resolved in sufficient detail to build an atomic model based on an AlphaFold prediction. Our results suggest a method by which flightin and myofilin cooperate to define the structure of the thick filament and explains a key myosin mutation that affects flightin incorporation. Drosophila is a genetic model organism for which our results can define strategies for functional testing. [ABSTRACT FROM AUTHOR]

Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in recent years, huge increase in the number of MPs solved via cryo-EM SPA at a resolution better than 3.0 Å in the Protein Data Bank (PDB) has been observed. However, sample preparation remains a significant challenge in the field. Here, we evaluated the MPs solved using cryo-EM SPA deposited in the PDB in the last two years at a resolution below 3.0 Å. The most critical parameters for sample preparation are as follows: (i) the surfactant used for protein extraction from the membrane, (ii) the surfactant, amphiphiles, nanodiscs or other molecules present in the vitrification step, (iii) the vitrification method employed, and (iv) the type of grids used. The aim is not to provide a definitive answer on the optimal sample conditions for cryo-EM SPA of MPs but rather assess the current trends in the MP structural biology community towards obtaining high-resolution cryo-EM structures. [ABSTRACT FROM AUTHOR]

Macromolecular assemblies, such as protein complexes, undergo continuous structural dynamics, including global reconfigurations critical for their function. Two fast analytical methods are widely used to study these global dynamics, namely elastic network model normal mode analysis and principal component analysis of ensembles of structures. These approaches have found wide use in various computational studies, driving the development of complex pipelines in several software packages. One common theme has been conformational sampling through hybrid simulations incorporating all-atom molecular dynamics and global modes of motion. However, wide functionality is only available for experienced programmers with limited capabilities for other users. We have, therefore, integrated one popular and extensively developed software for such analyses, the ProDy Python application programming interface, into the Scipion workflow engine. This enables a wider range of users to access a complete range of macromolecular dynamics pipelines beyond the core functionalities available in its command-line applications and the normal mode wizard in VMD. The new protocols and pipelines can be further expanded and integrated into larger workflows, together with other software packages for cryo-electron microscopy image analysis and molecular simulations. We present the resulting plugin, Scipion-EM-ProDy, in detail, highlighting the rich functionality made available by its development. [ABSTRACT FROM AUTHOR]

Single particle cryo-electron microscopy (cryo-EM) has emerged as the prevailing method for near-atomic structure determination, shedding light on the important molecular mechanisms of biological macromolecules. However, the inherent dynamics and structural variability of biological complexes coupled with the large number of experimental images generated by a cryo-EM experiment make data processing nontrivial. In particular, ab initio reconstruction and atomic model building remain major bottlenecks that demand substantial computational resources and manual intervention. Approaches utilizing recent innovations in artificial intelligence (AI) technology, particularly deep learning, have the potential to overcome the limitations that cannot be adequately addressed by traditional image processing approaches. Here, we review newly proposed AI-based methods for ab initio volume generation, heterogeneous 3D reconstruction, and atomic model building. We highlight the advancements made by the implementation of AI methods, as well as discuss remaining limitations and areas for future development. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Simple Summary: The treatment of B cell malignancies was transformed by the development of a monoclonal antibody—rituximab—that targets CD20, a protein expressed on the surface of B cells. However, some types of B cell malignancies do not express CD20 or can reduce the expression of the protein to escape therapy. Consequently, there is a need to develop tailored therapies against other targets expressed by B cells. The structures of key B cell proteins, the B cell receptor, and its co-receptors CD22, CD19 and CD81, have recently been solved and provide the opportunity to guide development of new antibodies and other therapies targeted at malignant B cells. Here, we review high-resolution protein structures of the BCR, CD22, CD19 and CD81 molecules, treatments that have been developed against these targets and discuss structural features that will enable the design of novel antibodies. B cells are central to the adaptive immune response, providing long lasting immunity after infection. B cell activation is mediated by a cell surface B cell receptor (BCR) following recognition of an antigen. BCR signaling is modulated by several co-receptors including CD22 and a complex that contains CD19 and CD81. Aberrant signaling through the BCR and co-receptors promotes the pathogenesis of several B cell malignancies and autoimmune diseases. Treatment of these diseases has been revolutionized by the development of monoclonal antibodies that bind to B cell surface antigens, including the BCR and its co-receptors. However, malignant B cells can escape targeting by several mechanisms and until recently, rational design of antibodies has been limited by the lack of high-resolution structures of the BCR and its co-receptors. Herein we review recently determined cryo-electron microscopy (cryo-EM) and crystal structures of the BCR, CD22, CD19 and CD81 molecules. These structures provide further understanding of the mechanisms of current antibody therapies and provide scaffolds for development of engineered antibodies for treatment of B cell malignancies and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Ribosome assembly is one of the most fundamental processes of gene expression and has served as a playground for investigating the molecular mechanisms of how protein–RNA complexes (RNPs) assemble. A bacterial ribosome is composed of around 50 ribosomal proteins, several of which are co-transcriptionally assembled on a ~4500-nucleotide-long pre-rRNA transcript that is further processed and modified during transcription, the entire process taking around 2 min in vivo and being assisted by dozens of assembly factors. How this complex molecular process works so efficiently to produce an active ribosome has been investigated over decades, resulting in the development of a plethora of novel approaches that can also be used to study the assembly of other RNPs in prokaryotes and eukaryotes. Here, we review biochemical, structural, and biophysical methods that have been developed and integrated to provide a detailed and quantitative understanding of the complex and intricate molecular process of bacterial ribosome assembly. We also discuss emerging, cutting-edge approaches that could be used in the future to study how transcription, rRNA processing, cellular factors, and the native cellular environment shape ribosome assembly and RNP assembly at large. [ABSTRACT FROM AUTHOR]

Heterogeneous three-dimensional (3D) reconstruction in single-particle cryo-electron microscopy (cryo-EM) is an important but very challenging technique for recovering the conformational heterogeneity of flexible biological macromolecules such as proteins in different functional states. Heterogeneous projection image classification is a feasible solution to solve the structural heterogeneity problem in single-particle cryo-EM. The majority of heterogeneous projection image classification methods are developed using supervised learning technology or require a large amount of a priori knowledge, such as the orientations or common lines of the projection images, which leads to certain limitations in their practical applications. In this paper, an unsupervised heterogeneous cryo-EM projection image classification algorithm based on autoencoders is proposed, which only needs to know the number of heterogeneous 3D structures in the dataset and does not require any labeling information of the projection images or other a priori knowledge. A simple autoencoder with multi-layer perceptrons trained in iterative mode and a complex autoencoder with residual networks trained in one-pass learning mode are implemented to convert heterogeneous projection images into latent variables. The extracted high-dimensional features are reduced to two dimensions using the uniform manifold approximation and projection dimensionality reduction algorithm, and then clustered using the spectral clustering algorithm. The proposed algorithm is applied to two heterogeneous cryo-EM datasets for heterogeneous 3D reconstruction. Experimental results show that the proposed algorithm can effectively extract category features of heterogeneous projection images and achieve high classification and reconstruction accuracy, indicating that the proposed algorithm is effective for heterogeneous 3D reconstruction in single-particle cryo-EM. [ABSTRACT FROM AUTHOR]

Alphaviruses are spherical, enveloped RNA viruses primarily transmitted by mosquitoes, and cause significant arthritogenic and neurotropic disease in humans and livestock. Previous reports have shown that—in contrast to prototypical icosahedral viruses—alphaviruses incorporate frequent defects, and these may serve important functions in the viral life cycle. We confirm the genus-wide pleomorphism in live viral particles and extend our understanding of alphavirus assembly through the discovery of an alternate architecture of Eastern equine encephalitis virus (EEEV) particles. The alternate T = 3 icosahedral architecture differs in triangulation number from the classic T = 4 icosahedral organization that typifies alphaviruses, but the alternate architecture maintains the quasi-equivalence relationship of asymmetric units. The fusion spike glycoproteins are more loosely apposed in the T = 3 form with corresponding changes in the underlying capsid protein lattice. This alternate architecture could potentially be exploited in engineering alphavirus-based particles for delivery of alphaviral or other RNA. [ABSTRACT FROM AUTHOR]

Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323–785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements. [ABSTRACT FROM AUTHOR]

Cryo-electron microscopy (cryo-EM) has become an unrivaled tool for determining the structure of macromolecular complexes. The biological function of macromolecular complexes is inextricably tied to the flexibility of these complexes. Single particle cryo-EM can reveal the conformational heterogeneity of a biochemically pure sample, leading to well-founded mechanistic hypotheses about the roles these complexes play in biology. However, the processing of increasingly large, complex datasets using traditional data processing strategies is exceedingly expensive in both user time and computational resources. Current innovations in data processing capitalize on artificial intelligence (AI) to improve the efficiency of data analysis and validation. Here, we review new tools that use AI to automate the data analysis steps of particle picking, 3D map reconstruction, and local resolution determination. We discuss how the application of AI moves the field forward, and what obstacles remain. We also introduce potential future applications of AI to use cryo-EM in understanding protein communities in cells. [ABSTRACT FROM AUTHOR]

Protein folding is the most fundamental and universal example of biomolecular self-organization and is characterized as an intramolecular process. In contrast, amyloidogenic proteins can interact with one another, leading to protein aggregation. The energy landscape of amyloid fibril formation is characterized by many minima for different competing low-energy structures and, therefore, is much more enigmatic than that of multiple folding pathways. Thus, to understand the entire energy landscape of protein aggregation, it is important to elucidate the full picture of conformational changes and polymorphisms of amyloidogenic proteins. This review provides an overview of the conformational diversity of amyloid-β (Aβ) characterized from experimental and theoretical approaches. Aβ exhibits a high degree of conformational variability upon transiently interacting with various binding molecules in an unstructured conformation in a solution, forming an α-helical intermediate conformation on the membrane and undergoing a structural transition to the β-conformation of amyloid fibrils. This review also outlines the structural polymorphism of Aβ amyloid fibrils depending on environmental factors. A comprehensive understanding of the energy landscape of amyloid formation considering various environmental factors will promote drug discovery and therapeutic strategies by controlling the fibril formation pathway and targeting the consequent morphology of aggregated structures. [ABSTRACT FROM AUTHOR]

The number of people living with multiple sclerosis (MS) in developed countries is increasing. The management of patients is hindered by the absence of reliable laboratory tests accurately reflecting the disease activity. Extracellular vesicles (EVs) of different cell origin were reportedly elevated in MS patients. We assessed the diagnostic potential, with flow cytometry analysis, of fresh large EVs (lEVs), which scattered more light than the 590 nm silica beads and were isolated from the blood plasma of relapsing remitting MS patients. Venous blood was collected from 15 patients and 16 healthy controls (HC). The lEVs were isolated from fresh platelet-free plasma by centrifugation, labelled with antibodies and the presence of platelet (CD41+, CD36+), endothelial (CD105+), erythrocyte (CD235a+), leukocyte (CD45+, CD19+, CD3+) and phosphatidylserine (Annexin V+) positive lEVs was analyzed using standard flow cytometry. Cryo-electron microscopy was used to verify the presence of EVs in the analyzed plasma fractions. MS patients experiencing acute relapse had slightly reduced relative levels (% of positive lEVs) of CD105+, CD45+, CD3+, CD45+CD3+ or CD19+ labelled lEVs in comparison to healthy controls. An analysis of other markers or a comparison of absolute lEV counts (count of lEVs/µL) did not yield any significant differences. Our data do not support the hypothesis that the exacerbation of the disease in RRMS patients leads to an increased numbers of circulating plasma lEVs which can be monitored by standard flow cytometry. [ABSTRACT FROM AUTHOR]

Dps (DNA-binding protein from starved cells) is well known for the structural protection of bacterial DNA by the formation of highly ordered intracellular assemblies under stress conditions. Moreover, this ferritin-like protein can perform fast oxidation of ferrous ions and subsequently accumulate clusters of ferric ions in its nanocages, thus providing the bacterium with physical and chemical protection. Here, cryo-electron microscopy was used to study the accumulation of iron ions in the nanocage of a Dps protein from Escherichia coli. We demonstrate that Fe2+ concentration in the solution and incubation time have an insignificant effect on the volume and the morphology of iron minerals formed in Dps nanocages. However, an increase in the Fe2+ level leads to an increase in the proportion of larger clusters and the clusters themselves are composed of discrete ~1–1.5 nm subunits. [ABSTRACT FROM AUTHOR]

Photosystem II is a light-driven water-plastoquinone oxidoreductase present in cyanobacteria, algae and plants. It produces molecular oxygen and protons to drive ATP synthesis, fueling life on Earth. As a multi-subunit membrane-protein-pigment complex, Photosystem II undergoes a dynamic cycle of synthesis, damage, and repair known as the Photosystem II lifecycle, to maintain a high level of photosynthetic activity at the cellular level. Cyanobacteria, oxygenic photosynthetic bacteria, are frequently used as model organisms to study oxygenic photosynthetic processes due to their ease of growth and genetic manipulation. The cyanobacterial PSII structure and function have been well-characterized, but its lifecycle is under active investigation. In this review, advances in studying the lifecycle of Photosystem II in cyanobacteria will be discussed, with a particular emphasis on new structural findings enabled by cryo-electron microscopy. These structural findings complement a rich and growing body of biochemical and molecular biology research into Photosystem II assembly and repair. [ABSTRACT FROM AUTHOR]

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species, with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a 3.3 Å cryo-EM structure of the TBEV virion of the Kuutsalo-14 isolate, confirming the overall organisation of the virus. We observe conformational switching of the peripheral and transmembrane helices of M protein, which can explain the quasi-equivalent packing of the viral proteins and highlights their importance in stabilising membrane protein arrangement in the virion. The residues responsible for M protein interactions are highly conserved in TBEV but not in the structurally studied Hypr strain, nor in mosquito-borne flaviviruses. These interactions may compensate for the lower number of hydrogen bonds between E proteins in TBEV compared to the mosquito-borne flaviviruses. The structure reveals two lipids bound in the E protein which are important for virus assembly. The lipid pockets are comparable to those recently described in mosquito-borne Zika, Spondweni, Dengue, and Usutu viruses. Our results thus advance the understanding of tick-borne flavivirus architecture and virion-stabilising interactions. [ABSTRACT FROM AUTHOR]

Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 [Fe4S4]-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM. [ABSTRACT FROM AUTHOR]

Mitochondrial cristae are dynamic invaginations of the inner membrane and play a key role in its metabolic capacity to produce ATP. Structural alterations caused by either genetic abnormalities or detrimental environmental factors impede mitochondrial metabolic fluxes and lead to a decrease in their ability to meet metabolic energy requirements. While some of the key proteins associated with mitochondrial cristae are known, very little is known about how the inner membrane dynamics are involved in energy metabolism. In this study, we present a computational strategy to understand how cristae are formed using a phase-based separation approach of both the inner membrane space and matrix space, which are explicitly modeled using the Cahn–Hilliard equation. We show that cristae are formed as a consequence of minimizing an energy function associated with phase interactions which are subject to geometric boundary constraints. We then extended the model to explore how the presence of calcium phosphate granules, entities that form in calcium overload conditions, exert a devastating inner membrane remodeling response that reduces the capacity for mitochondria to produce ATP. This modeling approach can be extended to include arbitrary geometrical constraints, the spatial heterogeneity of enzymes, and electrostatic effects to mechanize the impact of ultrastructural changes on energy metabolism. [ABSTRACT FROM AUTHOR]

Cryo-electron microscopy (cryo-EM) is a structure determination method for large molecular complexes. As more and more atomic structures are determined using this technique, it is becoming possible to perform statistical characterization of side-chain conformations. Two data sets were involved to characterize block lengths for each of the 18 types of amino acids. One set contains 9131 structures resolved using X-ray crystallography from density maps with better than or equal to 1.5 Å resolutions, and the other contains 237 protein structures derived from cryo-EM density maps with 2–4 Å resolutions. The results show that the normalized probability density function of block lengths is similar between the X-ray data set and the cryo-EM data set for most of the residue types, but differences were observed for ARG, GLU, ILE, LYS, PHE, TRP, and TYR for which conformations with certain shorter block lengths are more likely to be observed in the cryo-EM set with 2–4 Å resolutions. [ABSTRACT FROM AUTHOR]

PrPSc (scrapie isoform of the prion protein) prions are the infectious agent behind diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids (deer, elk, moose, and reindeer), as well as goat and sheep scrapie. PrPSc is an alternatively folded variant of the cellular prion protein, PrPC, which is a regular, GPI-anchored protein that is present on the cell surface of neurons and other cell types. While the structure of PrPC is well studied, the structure of PrPSc resisted high-resolution determination due to its general insolubility and propensity to aggregate. Cryo-electron microscopy, X-ray fiber diffraction, and a variety of other approaches defined the structure of PrPSc as a four-rung β-solenoid. A high-resolution structure of PrPSc still remains to be solved, but the four-rung β-solenoid architecture provides a molecular framework for the autocatalytic propagation mechanism that gives rise to the alternative conformation of PrPSc. Here, we summarize the current knowledge regarding the structure of PrPSc and speculate about the molecular conversion mechanisms that leads from PrPC to PrPSc. [ABSTRACT FROM AUTHOR]

To take advantage of recent advances in genomics and proteomics it is critical that the three-dimensional physical structure of biological macromolecules be determined. Cryo-Electron Microscopy (cryo-EM) is a promising and improving method for obtaining this data, however resolution is often not sufficient to directly determine the atomic scale structure. Despite this, information for secondary structure locations is detectable. De novo modeling is a computational approach to modeling these macromolecular structures based on cryo-EMderived data. During de novo modeling a mapping between detected secondary structures and the underlying amino acid sequence must be identified. DP-TOSS (Dynamic Programming for determining the Topology Of Secondary Structures) is one tool that attempts to automate the creation of this mapping. By treating the correspondence between the detected structures and the structures predicted from sequence data as a constraint graph problem DP-TOSS achieved good accuracy in its original iteration. In this paper, we propose modifications to the scoring methodology of DP-TOSS to improve its accuracy. Three scoring schemes were applied to DP-TOSS and tested: (i) a skeleton-based scoring function; (ii) a geometry-based analytical function; and (iii) a multi-well potential energy-based function. A test of 25 proteins shows that a combination of these schemes can improve the performance of DP-TOSS to solve the topology determination problem for macromolecule proteins. [ABSTRACT FROM AUTHOR]

We propose that viruses with geometric defects are not necessarily flawed viruses. A geometric defect may be a reactive site. Defects may facilitate assembly, dissociation, or accessibility of cellular proteins to virion components. In single molecule studies of hepadnavirus assembly, defects and overgrowth are common features. Icosahedral alphaviruses and flaviviruses, among others, have capsids with geometric defects. Similarly, immature retroviruses, which are non-icosahedral, have numerous "errors". In many viruses, asymmetric exposure of interior features allows for regulated genome release or supports intracellular trafficking. In these viruses, the defects likely serve a biological function. Commonly used approaches for spherical virus structure determination use symmetry averaging, which obscures defects. We suggest that there are three classes of asymmetry: regular asymmetry as might be found in a tailed phage, irregular asymmetry as found, for example, in defects randomly trapped during assembly, and dynamic asymmetry due to Brownian dynamics of virus capsids. Awareness of their presence and recent advances in electron microscopy will allow unprecedented investigation of capsid irregularities to investigate their biological relevance. [ABSTRACT FROM AUTHOR]

In the tailed bacteriophages, DNA is packaged into spherical procapsids, leading to expansion into angular, thin-walled mature capsids. In many cases, this maturation is accompanied by cleavage of the major capsid protein (CP) and other capsid-associated proteins, including the scaffolding protein (SP) that serves as a chaperone for the assembly process. Staphylococcus aureus bacteriophage 80α is capable of high frequency mobilization of mobile genetic elements called S. aureus pathogenicity islands (SaPIs), such as SaPI1. SaPI1 redirects the assembly pathway of 80α to form capsids that are smaller than those normally made by the phage alone. Both CP and SP of 80α are N-terminally processed by a host-encoded protease, Prp. We have analyzed phage mutants that express pre-cleaved or uncleavable versions of CP or SP, and show that the N-terminal sequence in SP is absolutely required for assembly, but does not need to be cleaved in order to produce viable capsids. Mutants with pre-cleaved or uncleavable CP display normal viability. We have used cryo-EM to solve the structures of mature capsids from an 80α mutant expressing uncleavable CP, and from wildtype SaPI1. Comparisons with structures of 80α and SaPI1 procapsids show that capsid maturation involves major conformational changes in CP, consistent with a release of the CP N-arm by SP. The hexamers reorganize during maturation to accommodate the different environments in the 80α and SaPI1 capsids. [ABSTRACT FROM AUTHOR]

All dsDNA phages encode two proteins involved in host lysis, an endolysin and a holin that target the peptidoglycan and cytoplasmic membrane, respectively. Bacteriophages that infect Gram-negative bacteria encode additional proteins, the spanins, involved in disruption of the outer membrane. Recently, a gene located in the lytic cassette was identified in the genomes of mycobacteriophages, which encodes a protein (LysB) with mycolyl-arabinogalactan esterase activity. Taking in consideration the complex mycobacterial cell envelope that mycobacteriophages encounter during their life cycle, it is valuable to evaluate the role of these proteins in lysis. In the present work, we constructed an Ms6 mutant defective on lysB and showed that Ms6 LysB has an important role in lysis. In the absence of LysB, lysis still occurs but the newly synthesized phage particles are deficiently released to the environment. Using cryo-electron microscopy and tomography to register the changes in the lysis phenotype, we show that at 150 min post-adsorption, mycobacteria cells are incompletely lysed and phage particles are retained inside the cell, while cells infected with Ms6wt are completely lysed. Our results confirm that Ms6 LysB is necessary for an efficient lysis of Mycobacterium smegmatis, acting, similarly to spanins, in the third step of the lysis process. [ABSTRACT FROM AUTHOR]

LuIII, a protoparvovirus pathogenic to rodents, replicates in human mitotic cells, making it applicable for use to kill cancer cells. This virus group includes H-1 parvovirus (H-1PV) and minute virus of mice (MVM). However, LuIII displays enhanced oncolysis compared to H-1PV and MVM, a phenotype mapped to the major capsid viral protein 2 (VP2). This suggests that within LuIII VP2 are determinants for improved tumor lysis. To investigate this, the structure of the LuIII virus-like-particle was determined using single particle cryo-electron microscopy and image reconstruction to 3.17 Å resolution, and compared to the H-1PV and MVM structures. The LuIII VP2 structure, ordered from residue 37 to 587 (C-terminal), had the conserved VP topology and capsid morphology previously reported for other protoparvoviruses. This includes a core β-barrel and α-helix A, a depression at the icosahedral 2-fold and surrounding the 5-fold axes, and a single protrusion at the 3-fold axes. Comparative analysis identified surface loop differences among LuIII, H-1PV, and MVM at or close to the capsid 2- and 5-fold symmetry axes, and the shoulder of the 3-fold protrusions. The 2-fold differences cluster near the previously identified MVM sialic acid receptor binding pocket, and revealed potential determinants of protoparvovirus tumor tropism. [ABSTRACT FROM AUTHOR]

Prior to the discovery of the mimivirus in 2003, viruses were thought to be physically small and genetically simple. Mimivirus, with its ~750-nm particle size and its ~1.2-Mbp genome, shattered these notions and changed what it meant to be a virus. Since this discovery, the isolation and characterization of giant viruses has exploded. One of the more recently discovered giant viruses, Samba virus, is a Mimivirus that was isolated from the Rio Negro in the Brazilian Amazon. Initial characterization of Samba has revealed some structural information, although the preparation techniques used are prone to the generation of structural artifacts. To generate more native-like structural information for Samba, we analyzed the virus through cryo-electron microscopy, cryo-electron tomography, scanning electron microscopy, and fluorescence microscopy. These microscopy techniques demonstrated that Samba particles have a capsid diameter of ~527 nm and a fiber length of ~155 nm, making Samba the largest Mimivirus yet characterized. We also compared Samba to a fiberless mimivirus variant. Samba particles, unlike those of mimivirus, do not appear to be rigid, and quasi-icosahedral, although the two viruses share many common features, including a multi-layered capsid and an asymmetric nucleocapsid, which may be common amongst the Mimiviruses. [ABSTRACT FROM AUTHOR]

p97/VCP, a highly conserved type II ATPase associated with diverse cellular activities (AAA+ ATPase), is an important therapeutic target in the treatment of neurodegenerative diseases and cancer. p97 performs a variety of functions in the cell and facilitates virus replication. It is a mechanochemical enzyme that generates mechanical force from ATP-binding and hydrolysis to perform several functions, including unfolding of protein substrates. Several dozens of cofactors/adaptors interact with p97 and define the multifunctionality of p97. This review presents the current understanding of the molecular mechanism of p97 during the ATPase cycle and its regulation by cofactors and small-molecule inhibitors. We compare detailed structural information obtained in different nucleotide states in the presence and absence of substrates and inhibitors. We also review how pathogenic gain-of-function mutations modify the conformational changes of p97 during the ATPase cycle. Overall, the review highlights how the mechanistic knowledge of p97 helps in designing pathway-specific modulators and inhibitors., Competing Interests: The authors declare no conflict of interest.

Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae. [ABSTRACT FROM AUTHOR]