HIV-1 virions harbor a core composed of a conical shell of capsid proteins that encases the viral ribonucleoprotein complex (vRNP). The vRNP is composed of HIV-1 genomic RNA (gRNA) and nucleocapsid (NC), reverse transcriptase (RT), and integrase (IN) proteins. Using a rapid affinity-capture purification approach, we have enriched for native HIV-1 capsids and analyzed the roles of IN-gRNA interactions and capsid lattice assembly on HIV-1 core formation. Following disruption of IN-gRNA interactions by allosteric IN inhibitor treatment or IN-deletion, we find that RT remains associated with cores that have effectively lost their IN, gRNA, and NC contents. Morphologically disrupted capsids formed by clinical inhibitor lenacapavir treatment or capsid hexamer cross-linking, by contrast, retained IN-gRNA but lost their RT and NC contents. These contrasting effects reveal that IN and the assembling capsid architecture cooperatively regulate the formation and encapsulation of vRNPs into the viral core during infectious HIV-1 morphogenesis.

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 front end 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.

Glaucoma is a group of neurodegenerative diseases that together are the leading cause of irreversible blindness worldwide. Myocilin-associated glaucoma is an inherited form of this disease, caused by intracellular aggregation of misfolded mutant myocilin. In vitro, the myocilin C-terminal olfactomedin domain (OLF), the relevant domain for glaucoma pathogenesis, can be driven to form amyloid-like fibrils under mild conditions. Here we characterize a species present during in vitro fibrillization. Purified OLF was subjected to fibrillization at concentrations required for downstream electron microscopy imaging and NMR spectroscopy. Additional biophysical techniques, including analytical ultracentrifugation and X-ray crystallography, were employed to further characterize the multicomponent mixture. Negative stain transmission electron microscopy (TEM) shows a non-native species reminiscent of known prefibrillar oligomers from other amyloid systems, NMR indicates a minor population of partially misfolded species is present in solution, and cryo-EM imaging shows two-dimensional protein arrays. The predominant soluble species remaining in solution after the fibril reaction is natively folded, as evidenced by X-ray crystallography. In summary, after incubating OLF under fibrillization-promoting conditions, there is a heterogeneous mixture consisting of soluble folded protein, mature amyloid-like fibrils, and partially misfolded intermediate species that at present belie additional molecular detail. The characterization of OLF fibrillar species illustrates the challenges associated with developing a comprehensive understanding of the fibrillization process for large, non-model amyloidogenic proteins.

Mitochondrial health relies on the membrane fission mediated by dynamin-related protein 1 (Drp1). Previous structural studies of Drp1 on remodeled membranes were hampered by heterogeneity, leaving a critical gap in the understanding of the mitochondrial fission mechanisms. Here we present a cryo-electron microscopy structure of full-length human Drp1 decorated on membrane tubules. Using the reconstruction of average subtracted tubular regions (RASTR) technique, we report that Drp1 forms a locally ordered lattice along the tubule without global helical symmetry. The filaments in the lattice are similar to dynamin rungs with conserved stalk interactions. Adjacent filaments are connected by GTPase domain interactions in a novel stacked conformation. We identified two states of the Drp1 lattice among the heterogenous dataset representing conformational changes around hinge 1. Additionally, we observed contact between Drp1 and membrane that can be assigned to the variable domain sequence. Together these structures revealed a putative mechanism by which Drp1 constricts mitochondria membranes in a stepwise, "ratchet" manner.

Tubular structures are ubiquitous in biological systems and have been a focal point of cryo-electron microscopy (cryo-EM) structural analysis since the technique’s inception. A critical step in processing tubular cryo-EM data is particle classification by diameter, as uniformity in diameter is a prerequisite for high-resolution three-dimensional reconstructions. Conventional methods rely on cross-correlation-based algorithms, which require prior knowledge to generate reference images, or iterative two-dimensional (2D) classification, that align and cluster particles into a predefined number of classes—a process that is both time-consuming and subjective. To address these limitations, we developed diameTR, a computational tool that rapidly determines tubular diameters in a prior knowledge-free and reference-free manner using GPU-accelerated processing on a per-particle basis. When applied to homogeneous datasets, diameTR yields narrow diameter distributions aligning closely with published values. For heterogeneous samples, it enables the separation of subsets with distinct diameters, validated by 2D averaging. Notably, diameTR identified an unreported smaller diameter subset of particles with new helical symmetry parameters in the previously published KpFtsZ-Monobody dataset. Collectively, diameTR represents a robust, efficient solution for diameter determination in tubular cryo-EM samples, eliminating the need for extensive human intervention while significantly accelerating processing.

Single-particle cryo-electron microscopy (cryo-EM) has become an essential tool in structural biology. However, automating repetitive tasks remains an ongoing challenge in cryo-EM data-set processing. Here, we present a platform-independent convolutional neural network (CNN) tool for assessing the quality of 2D averages to enable the automatic selection of suitable particles for high-resolution reconstructions, termed CryoSift. We integrate CryoSift into a fully automated processing pipeline using the existing cryosparc-tools library. Our integrated and customizable 2D assessment workflow enables high-throughput processing that accommodates experienced to novice cryo-EM users.

Protein misfolding is a widespread phenomenon that can result in the formation of protein aggregates, which are markers of various disease states, including Alzheimer's disease (AD). In AD, amyloid beta (A(3) peptides are key players in the disease's progression, particularly the 40- and 42- residue variants, A(340 and A(342. These peptides aggregate to form amyloid plaques and contribute to neuronal toxicity. Recent research has shifted attention from solely A(3 fibrils to also include A(3 protofibrils and oligomers as potentially critical pathogenic agents. Particularly, oligomers demonstrate more significant toxicity compared to other A(3 specie. Hence, there is an increased interest in studying the correlation between toxicity and their structure and aggregation pathway. The present study investigates the aggregation of a 150 kDa A(342 oligomer that does not lead to fibril formation. Using negative stain transmission electron microscopy (TEM), size exclusion chromatography (SEC), dynamic light scattering (DLS), and cryo-electron microscopy (cryo-EM), we demonstrate that 150 kDa A(342 oligomers form higher-order string-like assemblies over time. These strings are unique from the classical A(3 fibrils. The significance of our work lies in elucidating molecular behavior of a novel non-fibrillar form of A(342 aggregate.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples. Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation. His-tagged specimens of interest are added to the tubules, and they can be frozen by conventional plunge freezing. We show that the nanotubes protect samples from the air/water interface and promote a wider range of orientations. The reconstruction of average subtracted tubular regions (RASTR) method allows for the removal of the nanotubule signal from the cryo-EM images resulting in isolated images of specimens of interest. Testing with β-galactosidase validates the method’s ability to capture particles at lower concentrations, overcome preferred orientations, and achieve near-atomic resolution reconstructions. Since the nanotubules can be identified and targeted automatically at low magnification, the method enables fully automated data collection. Furthermore, the particles on the tubes can be automatically identified and centered using 2D classification enabling particle picking without requiring prior information. Altogether, our approach that we call specimen preparation on a tube RASTR (SPOT-RASTR) holds promise for overcoming air-water interface and preferred orientation challenges and offers the potential for fully automated cryo-EM data collection and structure determination.

We have developed new tools for cryo-EM specimen preparation and data processing. With the rapid development of new algorithms for processing cryo-EM data, there is a need for a central platform for deploying and comparing the different algorithms. Magellon is a new platform that we have developed for cryo-EM data visualization as well as facile deployment and comparison of data processing algorithms. Magellon Viewer is web-based and is backed by a relational database for robust and straightforward data sharing and transfer of metadata between different sites. We will also discuss a new specimen preparation technique called specimen preparation on a tube with reconstruction of average subtracted tubular regions (SPOT-RASTR). SPOT-RASTR captures single particle specimens on a membrane nanotube. We show that the nanotubes, which are computationally erased, hold the specimens away from the air/water interface and promote full coverage of Euler space for single particle analysis. Our latest results on Magellon and SPOT-RASTR will be discussed.

Pathological variants in Trk-fused gene (TFG) have been implicated in a variety of neurodegenerative conditions. In particular, mutations within its amino-terminal PB1 domain have been suggested to cause hereditary spastic paraplegia (HSP), resulting in progressive lower limb spasticity and weakness. The structural basis for this effect is unknown. Here, we combine X-ray crystallography and cryo-electron microscopy to determine a structural model of TFG, demonstrating the mechanism by which it forms octameric ring complexes. A network of electrostatic and hydrophobic interactions defines the interface between protomers. Moreover, we show that mutations identified previously in HSP patients disrupt this interface, destabilizing octamers, which ultimately leads to axonopathy. Surprisingly, the impacts of these variants are not equivalent in vivo, highlighting the existence of multiple, distinct mechanisms by which TFG mutations contribute to neurodegenerative disease.

Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been developed by Direct Electron. The Apollo uses a novel event-based MAPS detector custom designed for ultra-fast electron counting. We have evaluated this new camera, finding that it delivers high detective quantum efficiency (DQE) and low coincidence loss, enabling high-quality electron counting data acquisition at up to nearly 80 input electrons per pixel per second. We further characterized the performance of Apollo for single particle cryo-EM on real biological samples. Using mouse apoferritin, Apollo yielded better than 1.9 Å resolution reconstructions at all three tested dose rates from a half-day data collection session each. With longer collection time and improved specimen preparation, mouse apoferritin was reconstructed to 1.66 Å resolution. Applied to a more challenging small protein aldolase, we obtained a 2.24 Å resolution reconstruction. The high quality of the map indicates that the Apollo has sufficiently high DQE to reconstruct smaller proteins and complexes with high-fidelity. Our results demonstrate that the Apollo camera performs well across a broad range of dose rates and is capable of capturing high quality data that produce high-resolution reconstructions for large and small single particle samples.

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.

Protein transport between the membranous compartments of the eukaryotic cells is mediated by the constant fission and fusion of the membrane-bounded vesicles from a donor to an acceptor membrane. While there are many membrane remodelling complexes in eukaryotes, COPII, COPI, and clathrin-coated vesicles are the three principal classes of coat protein complexes that participate in vesicle trafficking in the endocytic and secretory pathways. These vesicle-coat proteins perform two key functions: deforming lipid bilayers into vesicles and encasing selective cargoes. The three trafficking complexes share some commonalities in their structural features but differ in their coat structures, mechanisms of cargo sorting, vesicle formation, and scission. While the structures of many of the proteins involved in vesicle formation have been determined in isolation by X-ray crystallography, elucidating the proteins' structures together with the membrane is better suited for cryogenic electron microscopy (cryo-EM). In recent years, advances in cryo-EM have led to solving the structures and mechanisms of several vesicle trafficking complexes and associated proteins.

In the past decade, many developments have advanced the cryo-EM field in imaging technologies and processing methodologies. Still, there are many challenging samples that cannot be structurally characterized with conventional cryo-EM preparation procedures because they cannot be preserved natively in vitreous ice with traditional plunge freezing or, in some cases, even microfluidic assisted spray-plunge freezing. These recalcitrant specimens can touch the air-water interface, aggregate, or bind to the support film rather than remain suspended in the film's holes. Also, other problems that limit image analysis, like adopting a preferred orientation, might take place during the preparation of some samples. In this study, we adapted different alternative methods including streptavidin monolayer affinity supports and graphene solid supports using a known test specimen to systematically analyze their efficacy, reproducibility, and potential for high-resolution image analysis. Streptavidin affinity grids are made by forming a 2D crystal of streptavidin on a biotinylated lipid bed to which lightly biotinylated protein can bind to keep the protein stable and away from the surface. The graphene grid concept is similar, but the bed is a single layer of carbon atoms. For both, we produce grids and test specimens in-house. We compared these to commercial grids such as graphene, graphene oxide, and cryo well grids and found the in-house grids perform better. We also applied these alternative preservation techniques to a set of challenging samples that we wish to study but are hindered due to the above-mentioned limitations. Thus, our strategy to address the inherent limitations in plunge freezing recalcitrant specimens will be to pursue an affinity capture strategy.

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV’s molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near universal entry receptor, AAVR, and structures by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now β-strand A is the minor conformer and, for the major conformer, partially ordered N-termini near the 2-fold axis join the canonical capsid jellyroll fold at the βA-βB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCE With 150 clinical trials for 30 diseases underway, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction, has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.