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Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S . epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 Å density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S . epidermidis Cas10-Csm structure and the well-resolved bacterial (S . thermophilus ) and archaeal ( T . onnurineus ) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes.

The brains of Alzheimer’s disease (AD) patients contain numerous amyloid plaques that are diagnostic of the disease. The plaques are primarily composed of the amyloidogenic peptides proteins Aβ40 and Aβ42, which are derived by the processing of the amyloid pre-cursor protein (APP) by two proteases called β-secretase and γ-secretase. Aβ42 differs from Aβ40 in having two additional hydrophobic amino acids, ILE and ALA, at the C-terminus. A small percentage of AD is autosomal dominant (ADAD) and linked either to the genes for the presenilins, which are part of γ-secretase, or APP. Because ADAD shares most pathogenic features with widespread late-onset AD, Aβ peptides have become the focus of AD research. Fibrils formed by the aggregation of these peptides are the major component of plaques and were initially targeted in AD therapy. However, the fact that the abundance of plaques does not correlate well with cognitive decline in AD patients has led investigators to examine smaller Aβ aggregates called oligomers. The low levels and heterogeneity of Aβ oligomers have made the determination of their structures difficult, but recent structure determinations of oligomers either formed or initiated in detergents have been achieved. We report here on the structures of these oligomers and suggest how they may be involved in AD.

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans -translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans -translation by these molecules. These results show that trans -translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways. Antibiotic-resistant bacterial pathogens pose a substantial threat to human health. Here, aided by structural analyses, the authors describe the molecular mechanism behind the activity of a series of compounds that inhibit trans-translation and are effective in eradicating N. gonorrhoeae infection in mice.

Adeno-associated virus (AAV) vectors are preeminent in emerging clinical gene therapies. Generalizing beyond the most tractable genetic diseases will require modulation of cell specificity and immune neutralization. Interactions of AAV with its cellular receptor, AAVR, are key to understanding cell-entry and trafficking with the rigor needed to engineer tissue-specific vectors. Cryo-electron tomography shows ordered binding of part of the flexible receptor to the viral surface, with distal domains in multiple conformations. Regions of the virus and receptor in close physical proximity can be identified by cross-linking/mass spectrometry. Cryo-electron microscopy with a two-domain receptor fragment reveals the interactions at 2.4 Å resolution. AAVR binds between AAV’s spikes on a plateau that is conserved, except in one clade whose structure is AAVR-incompatible. AAVR’s footprint overlaps the epitopes of several neutralizing antibodies, prompting a re-evaluation of neutralization mechanisms. The structure provides a roadmap for experimental probing and manipulation of viral-receptor interactions.

Single-particle cryo-electron microscopy (cryo-EM) has revolutionized structural biology by enabling high-resolution determination of macromolecular structures. However, the field faces challenges in data management, processing workflow integration and software extensibility. We present Magellon , an innovative cryo-EM software platform that addresses these challenges through a modern microservices architecture. Magellon consists of an extensible backend with a web based frontend that we call Magellon Viewer . Together, these combine high-performance computing capabilities with an intuitive user interface, enabling researchers to efficiently process and analyze cryo-EM data. The platform's distinguishing features include a plugin based architecture, distributed processing capabilities, comprehensive monitoring systems, and a novel approach to data organization and visualization. A key philosophy of the approach is that the Magellon backend provides a platform that uses robust industry-standard libraries to orchestrate computational tasks while offering users and developers flexibility in selecting the computational resources for performing calculations. Magellon represents a significant advancement in cryo-EM software infrastructure, offering flexibility, scalability and extensibility while maintaining ease of use.

The COPII cage, formed by Sec13 and Sec31, organizes other proteins into a lattice on the endoplasmic-reticulum membrane and is involved in transporting cargo from the endoplasmic reticulum to the Golgi apparatus. A combination of cryo-EM and H/D-exchange MS analyses leads to a 12-Å-resolution model of the COPII cage, yielding insight into its architecture and assembly. COPII vesicles transport proteins from the endoplasmic reticulum to the Golgi apparatus. Previous COPII-cage cryo-EM structures lacked the resolution necessary to determine the residues of Sec13 and Sec31 that mediate assembly and flexibility of the COPII cage. Here we present a 12-Å structure of the human COPII cage, where the tertiary structure of Sec13 and Sec31 is clearly identifiable. We employ this structure and a homology model of the Sec13–Sec31 complex to create a reliable pseudoatomic model of the COPII cage. We combined this model with hydrogen/deuterium-exchange MS analysis to characterize four distinct contact regions at the vertices of the COPII cage. Furthermore, we found that the two-fold symmetry of the Sec31 dimeric region in Sec13–Sec31 is broken upon cage formation and that the resulting hinge is essential to form the proper edge geometry in COPII cages.

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.

Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is a hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape appears to have been the primary driver of AAV-DJ's directed evolution. Fondaparinux is an analog of cell surface heparan sulfate to which several AAVs bind during entry. Fondaparinux interacts with viral arginines at a known heparin binding site, without the large conformational changes whose presence was controversial in low-resolution imaging of AAV2-heparin complexes. The glycan density suggests multi-modal binding that could accommodate sequence variation and multivalent binding along a glycan polymer, consistent with a role in attachment, prior to more specific interactions with a receptor protein mediating entry.

Key Points Endoplasmic reticulum (ER) export by the coat protein complex-II (COPII) machinery involves the transport of nearly a third of the proteins that are encoded by the eukaryotic genome. The wide range of protein-folding stabilities that are accommodated by the COPII machinery indicates that the current view of the ER as a quality-control device should now be tempered with the more general principle that the energetics of the protein fold, independently of the final functional fold, directs ER export under physiological conditions. Cargo selection by the Sec23–Sec24 adaptor protein complex involves a high valency cargo adaptor platform. Through its binding to this cargo adaptor platform, cargo has an important role in directing vesicle formation and membrane traffic. The self-assembly properties of the Sec13–Sec31 complex, which result in the formation of a highly flexible cuboctahedral cage, direct the concentration of cargo into budding vesicles and coordinate cargo recruitment with membrane curvature and fission. The GTPase Sar1, which is involved in Sec23–Sec24 membrane recruitment during the early steps of coat protein complex (CPC) assembly, also has an important role in late events — it directs vesicle fission. Vesicle release that is mediated by Sar1–GTP hydrolysis is likely to be coordinated with the function of lipid-remodelling factors. The structural and biochemical properties that are involved in the function of the COPII export machinery combined with those that have been observed for cargo selection and the generation of coat complexes by clathrin-mediated pathways now provide a unifying model for CPC assembly in eukaryotic cells. The assembly of CPCs is driven by various kinetic parameters that control the individual steps of protein folding, cargo selection and collection, and membrane curvature and fission dynamics. These complexes are therefore highly versatile and robust trafficking machineries. CPCs — which include the COPII, clathrin and COPI coats — probably evolved from the more flexible cage structure that is found in the COPII-based pathway, which directs ER export, to the more selective and rigid clathrin-based and COPI-based cage structures, which are involved in post-ER trafficking pathways. Recent studies of the early secretory pathway have analysed cargo selection and transport-carrier formation by components of the endoplasmic-reticulum-associated coat protein complex-II (COPII). Results are indicative of a unifying model of cage and coat function in vesicle and tubule formation as well as fission in endomembrane traffic. Communication between compartments of the exocytic and endocytic pathways in eukaryotic cells involves transport carriers — vesicles and tubules — that mediate the vectorial movement of cargo. Recent studies of transport-carrier formation in the early secretory pathway have provided new insights into the mechanisms of cargo selection by coat protein complex-II (COPII) adaptor proteins, the construction of cage-protein scaffolds and fission. These studies are beginning to produce a unifying molecular and structural model of coat function in the formation and fission of vesicles and tubules in endomembrane traffic.

Endomembranes of eukaryotic cells are dynamic structures that are in continuous communication through the activity of specialized cellular machineries 1 , such as the coat protein complex II (COPII), which mediates cargo export from the endoplasmic reticulum (ER) 2 , 3 . COPII consists of the Sar1 GTPase, Sec23 and Sec24 (Sec23/24), where Sec23 is a Sar1-specific GTPase-activating protein and Sec24 functions in cargo selection, and Sec13 and Sec31 (Sec13/31), which has a structural role 3 . Whereas recent results have shown that Sec23/24 and Sec13/31 can self-assemble to form COPII cage-like particles 4 , we now show that Sec13/31 can self-assemble to form minimal cages in the absence of Sec23/24. We present a three-dimensional reconstruction of these Sec13/31 cages at 30 Å resolution using cryo-electron microscopy and single particle analysis. These results reveal a novel cuboctahedron geometry with the potential to form a flexible lattice and to generate a diverse range of containers. Our data are consistent with a model for COPII coat complex assembly in which Sec23/24 has a non-structural role as a multivalent ligand localizing the self-assembly of Sec13/31 to form a cage lattice driving ER cargo export.