Biology subjects

Stagg, S. M.

Publications and source records attributed to Stagg, S. M..

12 recordsLinked to original sources

bioRxiv2026

Stimulating microglial neuroinflammation with amyloid-beta oligomers compatible with solid-state NMR structural characterization

Soluble amyloid-{beta} (A{beta}) oligomers are considered the primary neurotoxic species in Alzheimer's disease, yet the relationship between oligomer structure and biological activity remains poorly understood. Oligomer sample preparations amenable to high-resolution structural characterization have not been tested in cell-based assays, mostly because detergents used to drive oligomer formation are toxic to cells. Here, we describe a detergent-free protocol in which size-exclusion chromatography-isolated A{beta}42 monomers spontaneously oligomerize at 37 {degrees}C in a concentration- and temperature-dependent manner. Biophysical characterization shows a {beta}-sheet structure by circular dichroism, a self-associating population above 100 kDa by silver-stained SDS-PAGE, and bead-like strings by negative-stain TEM resembling the previously characterized SDS-induced 150 kDa oligomer. Solid-state NMR spectroscopy of selectively 13C/15N-labeled 37 {degrees}C oligomers yields well-resolved spectra with {beta}-strand chemical shifts, and two-dimensional 13C-13C DARR and CHHC experiments provide initial inter-residue distance constraints that reveal both similarities to and differences from the 150 kDa oligomer. Treatment of mouse microglial cells (MMCs) with 50 nM 37 {degrees}C oligomers elicited a robust pro-inflammatory cytokine response that exceeded that of monomeric A{beta}42, as demonstrated by Luminex multiplex profiling and principal component analysis. hiPSC-derived microglia (iMGs) treated with 1 {micro}M oligomers also separated from vehicle, establishing activity in a human cellular model. Monomeric A{beta}42 was inactive in both systems, indicating that inflammatory activity depends on the assembly state of A{beta}42 rather than on the presence of peptide alone. These results establish a detergent-free A{beta}42 oligomer preparation that is simultaneously compatible with solid-state NMR structural characterization and biologically active in neuroinflammation assays, providing a platform for studying microglial immune responses to oligomers.

neuroscience↗

bioRxiv2026

Cryo-EM Reveals VP1/VP2 N-Terminal Conformations in Adeno-Associated Virus 2 Capsids.

The AAV2 capsid assembles from three viral proteins, VP1, VP2, and VP3, expressed from a single cap gene. In the assembled T=1 60-subunit icosahedral capsid, they have a canonical stoichiometry of 1:1:10. Relative to VP3, VP1 and VP2 have N-terminal extensions that are required for endosomal escape and nuclear trafficking; however, these regions remain poorly resolved due to low stoichiometry, symmetry mismatches, and conformational heterogeneity of their N-termini. Here, we generated empty AAV2 capsids using a baculovirus expression system with time-course harvests, yielding approximately 2:3:5 and 1:2:7. VP1:VP2:VP3 ratios on days 3 and 5 post-infection, respectively. Cryo-EM single-particle and subparticle reconstructions revealed distinct N-terminal configurations associated with these different VP stoichiometries. Day 3 capsids contained an internal density protruding from the twofold axis and lacked the ordered {beta}A preceding residue 237, whereas day 5 capsids lacked the twofold protrusion and instead showed density associated with the fivefold axis, with {beta}A ordered to approximately residues 219-221. Fivefold subparticle reconstruction of day 5 capsids further resolved a density plug occluding the pore and an underlying density ring. These findings link VP stoichiometry to distinct conformations of the VP1/VP2 N-termini and provide structural evidence for alternative organization of these regions within the AAV2 capsid, offering new insights into the conformational landscape of AAV capsid heterogeneity.

biophysics↗

bioRxiv2025

Magellon - an extensible platform for cryo-EM data visualization, management, and processing

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 platforms 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.

biophysics↗

bioRxiv2024

Aggregation Dynamics of a 150 kDa Aβ42 Oligomer: Insights from Cryo Electron Microscopy and Multimodal Analysis

I.Protein misfolding is a widespread phenomenon that can result in the formation of protein aggregates, which are markers of various disease states, including Alzheimers disease (AD). In AD, amyloid beta (A{beta}) peptides, particularly A{beta}40 and A{beta}42, are key players in the diseases progression, as they aggregate to form amyloid plaques and contribute to neuronal toxicity. Recent research has shifted attention from solely A{beta} fibrils to also include A{beta} protofibrils and oligomers as potentially critical pathogenic agents. Particularly, oligomers demonstrate greater toxicity compared to other A{beta} 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{beta}42 oligomer that does not lead to fibril formation over time. 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{beta}42 oligomers form higher-order string-like assemblies over time. The strings are unique from the classical A{beta} fibril structures. The significance of our work lies in elucidating molecular behavior of a novel non-fibrillar form of A{beta}42 aggregate.

biophysics↗

bioRxiv2022

Structural Model for Self-Limiting β-strand Arrangement Within an Alzheimer's Amyloid-β Oligomer

Previous reports revealed that sodium dodecyl sulfate near its critical micelle concentration can drive the assembly of A{beta}42 along an oligomeric pathway. This pathway produces a 150 kDa peptide oligomer (approximately 32 peptide molecules or protomers) that does not aggregate further into amyloid fibrils. Solid-state nuclear magnetic resonance (NMR) spectroscopy revealed structural features distinguishing the 150 kDa oligomer from fibrils. A puzzling feature was the coexistence of parallel and antiparallel {beta}-sheets within the oligomer structure. Here we present new atomic-level structural constraints obtained via solid-state NMR spectroscopy, benefitting from improved resolution via sample concentration by ultracentrifugation. In addition, two-dimensional cryo-electron microscopy (cryo-EM) reconstruction revealed a 4-fold symmetric shape. We propose a structural model to rationalize the solid-sate NMR- and cryo-EM-derived structural constraints. This model has a hollow square cylinder shape, with antiparallel {beta}-sheets formed by residues 33-39 lining the inner walls and parallel {beta}-sheets formed by residues 11-22 lining the outer walls. Within successive layers, the outer {beta}-strands on each side of the square cylinder alternate between two forms: one within a U-shaped protomer and another within L-shaped protomer. Molecular dynamics simulations show that, when the oligomer model is embedded in a lipid membrane, ions permeate through the central pore, with cation selectivity. The model further motivates an assembly pathway-based interpretation that may explain why the 150 kDa oligomer does not undergo further aggregation into amyloid fibrils. Significance StatementA{beta} oligomers are thought to be the most toxic species in Alzheimers disease. Their sizes range from 2 to [~]50 protomers. Most published experimental data on A{beta} oligomers indicate that they, like fibrils, are composed of {beta}-sheets, but it is a mystery why any {beta}-sheet aggregate would exist as a stable oligomer without undergoing further aggregation into fibrils. Here, structural constraints from solid-state NMR and cryo-EM led us to an oligomer model with a hollow square cylinder shape capable of conducting ions when embedded in a lipid membrane. Based on the model, we argue that geometric frustration may distinguish the assembly pathway that produces this oligomer from fibril-forming assembly pathways.

biochemistry↗

bioRxiv2022

The structure of a Type III-A CRISPR-Cas effector complex reveals conserved and idiosyncratic contacts to target RNA and crRNA among Type III-A systems

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 [A] 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.

molecular biology↗

bioRxiv2022

Characterizing the Resolution and Throughput of the Apollo Direct Electron Detector

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 [A] 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 [A] resolution. Applied to a more challenging small protein aldolase, we obtained a 2.24 [A] 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.

biochemistry↗

bioRxiv2022

Adeno-associated Virus Receptor-binding: Flexible Domains and Alternative Conformations through cryo-Electron Tomography of AAV2 and AAV5 complexes

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 AAVs 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 {beta}-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 {beta}A-{beta}B 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. IMPORTANCEWith 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.

microbiology↗

bioRxiv2020

Ribosome rescue inhibitors clear Neisseria gonorrhoeae in vivo using a new mechanism

The trans-translation pathway for rescuing stalled ribosomes is conserved and essential in bacterial pathogens but has no mammalian homolog, making it an ideal target for new antibiotics. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, resulting in broad-spectrum antibiotic activity. Optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles produced MBX-4132, which cleared multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Cryo-EM studies of non-stop ribosomes showed that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein L27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. One Sentence SummaryRibosome rescue inhibitors reveal a new conformation of the ribosome and kill drug-resistant Neisseria gonorrhoeae in vivo.

microbiology↗

bioRxiv2020

The structures of natively assembled clathrin coated vesicles

Vesicle trafficking by clathrin coated vesicles (CCVs) is one of the major mechanisms by which proteins and nutrients are absorbed by the cell and transported between organelles. The individual proteins comprising the coated vesicles include clathrin heavy chain, clathrin light chain, and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor {beta}2-appendages crosslink adjacent CHC {beta}-propellers and that the appendage densities reside almost exclusively in CCV hexagonal faces. We resolve how AP2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring {beta}-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

biophysics↗

bioRxiv2019

Reconstruction of Average Subtracted Tubular Regions (RASTR) Enables Structure Determination of Tubular Filaments by Cryo-EM

As the field of electron microscopy moves forward, the increasing complexity of samples being produced demand more involved processing methods. In this study, we have developed a new processing method for generating 3D reconstructions of tubular structures. Tubular biomolecules are common throughout many cellular processes and are appealing targets for a variety of biophysical research. Processing of tubules with helical symmetry is relatively straightforward for electron microscopy if the helical parameters are known, but tubular structures that deviate from helical symmetry (asymmetrical components, local but no global order, etc) present myriad issues. Here we present a new processing technique called Reconstruction of Average Subtracted Tubular Regions (RASTR), which was developed to reconstruct tubular structures without applying symmetry. We explain the RASTR approach and quantify its performance using three examples: a simulated symmetrical tubular filament, a symmetrical tubular filament from cryo-EM data, and a membrane tubule coated with locally ordered but not globally ordered proteins.

biophysics↗

bioRxiv2019

Throughput and Resolution with a Next Generation Direct Electron Detector

Direct electron detectors (DEDs) have revolutionized cryo-electron microscopy by facilitating correction of beam-induced motion and radiation damage and by providing high-resolution image capture. A new generation of DEDs has been developed by Direct Electron, the DE64, that has good performance in both integrating and counting modes. Integrating mode is superior in throughput while counting mode is superior in image quality. We show that despite being ~10x slower in throughput, counting mode is superior in terms of reconstruction resolution per unit time of data collection.

biophysics↗