Eosinophils (EOS) are white blood cells named for large granules that stain with the acidic dye eosin. Upon activation, EOS release basic cytotoxic proteins from these granules, including major basic protein (MBP-1). Traditional EM techniques indicate that MBP-1 forms a pseudocryalline core within each granule. Here, we applied cryo-focused ion beam (cryo-FIB) milling and montage cryo-electron tomography (MPACT) to study donor-derived human EOS. We demonstrated that the granule core is a three-dimensional (3D) protein nanocrystal and determined its native-state lattice organization using micro-electron diffraction (micro-ED). We noted double-membrane vesicles hugging granules of activated EOS. These have the dimensions of “Sombrero vesicles” noted by traditional EM and thought to mediate “piecemeal degranulation.” Unexpectedly, we also found free crystalline cores along cell peripheries. Thus, combining the strengths of cryo-FIB milling, cryo-ET, and micro-ED, we characterized the in-situ structure of EOS MBP-1 and the surrounding macromolecules within the native cellular landscape. The fresh observations raise new questions about release of granule contents from EOS.

Since the outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV), a virus that has caused a high case fatality rate of 36%, other merbecoviruses have been reported to also be capable of infecting human cells. Given the threat of Merbecovirus spillover to humans, we developed virus-like particle vaccines presenting the S2 subunit proteins of MERS-CoV, NeoCoV, HKU4, or HKU25. Mice were vaccinated with the homotypic vaccines, and IgG endpoint titers were measured against S2 proteins of the same panel of viruses, confirming high cross-reactivity across all four viruses. Based on characterization by antigenic cartography, MERS-CoV and HKU4 S2 proteins were selected as optimal components for a cocktail vaccine. MERS-CoV and NeoCoV homotypic vaccines, along with the mixture vaccine, provided partial protection in transgenic mice against a MERS-CoV challenge. These findings could serve as an important step toward designing pan-Merbecovirus vaccines in preparation for future outbreaks.

The accumulation of Alpha-synuclein (Asyn) fibrils is the defining pathologic feature in Parkinson Disease (PD), Lewy Body Dementia (LBD), and Multiple System Atrophy (MSA). As such, the process of Asyn fibril formation has been an important research area and fibrils themselves have become attractive targets for disease diagnosis and therapeutic intervention. Due to the presence of mixed populations of fibrillar proteins associated with neurodegenerative diseases in brain tissue, high-resolution structures of Asyn fibrils are essential for the design of high-specificity imaging and therapeutic agents. Approximately one hundred high-resolution solid-state NMR (SSNMR) spectroscopy and cryo-electron microscopy (cryo-EM) structures of Asyn fibrils have been deposited to the Protein Databank (PDB); intriguingly there is significant polymorphism among them. Understanding the molecular makeup and characteristic features of each structural polymorph can determine conserved structural motifs which can be used as templates to design ligands with high specificity for clinical use. Utilizing standard alignment tools and density-based clustering approaches, we objectively classify fibril structures by tertiary structure type. We find that 81% of the structures cluster into two polymorph classes. Within each class, additional subtle variations are observed which position sidechains in specific, conserved orientations, well poised as druggable targets. Furthermore, we find that the conserved structural motifs associated with each class are found in all but one published Asyn fibril structure. We consider these classifications and conserved motifs in the context of disease-relevant fibril structures and offer a perspective on the utility of in vitro fibrils as substrates for drug development and models for disease pathogenesis.

The physiological role of a-synuclein (a-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, a-syn will self-assemble into beta-sheet-rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient-derived brain tissues have concluded that these inclusions are associated with Parkinson's disease, the second most common neurodegenerative disorder, and other synuclein-related diseases called synucleinopathies. In addition, repetitions of specific mutations to the SNCA gene, the gene that encodes a-syn, result in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of a-syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 & Aring;. Here, we provide a protocol for a-syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step-by-step protocol for high-resolution cryo-EM structure determination of a-syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high-resolution cryo-EM map. This workflow resulted in a 2.04 & Aring; structure of a-syn fibrils with excellent resolution of residues 36-97 and an additional island of density for residues 15-22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of a-syn and other amyloid fibrils.

The continuing emergence of immune evasive SARS-CoV-2 variants and the previous SARS-CoV-1 outbreak collectively underscore the need for broadly protective sarbecovirus vaccines. Targeting the conserved S2 subunit of SARS-CoV-2 is a particularly promising approach to elicit broad protection. Here, we describe a nanoparticle vaccine displaying multiple copies of the SARS-CoV-1 S2 subunit. This vaccine alone, or as a cocktail with a SARS-CoV-2 S2 subunit vaccine, protects female transgenic K18-hACE2 mice from challenges with Omicron subvariant XBB as well as several sarbecoviruses identified as having pandemic potential including the bat sarbecovirus WIV1, BANAL-236, and a pangolin sarbecovirus. Challenge studies in female Fc-γ receptor knockout mice reveal that antibody-based cellular effector mechanisms play a role in protection elicited by these vaccines. These results demonstrate that our S2-based vaccines provide broad protection against clade 1 sarbecoviruses and offer insight into the mechanistic basis for protection. Understanding the induced and cross reactive immunity to sarbecoviruses is an important step in rationale and widely applicable vaccine design. Here the authors use a multivalent S2 subunit vaccine and demonstrate protection in female mice against SARS-CoV-2-like and SARS-CoV-1-like coronaviruses.

We present a novel microfluidic flow cell, or Flow-enabled Light and Ultrastructural Imaging Device for Correlative Electron and Light Localization (FLUID-CELL), that connects fluorescence light microscopy (FLM) and cryo-electron microscopy (cryo-EM) for advanced biological imaging and ultrastructural and structural analyses. The design of the FLUID-CELL features a precisely engineered microchannel that maintains native cell culturing conditions, supporting correlation and enabling real-time observation by FLM, as well as subsequent cryo-EM analysis. In this study, this device enabled practical FLM imaging over extended experimental periods, consistent sample handling, and the ability to perform correlative imaging. This capability connects dynamic cellular events imaged by fluorescence light microscopy with high-resolution ultrastructural data collected with cryo-EM. Our dual-modality approach streamlines the workflow and opens new possibilities for investigating the relationship between cellular function and molecular architecture at the nanoscale.

Fibrillar aggregation of α-synuclein (αSyn) is a hallmark of Parkinson's disease (PD) and related disorders, including multiple system atrophy (MSA) and dementia with Lewy bodies (DLB). Despite advances in αSyn fibril structural characterization, the relevance of in vitro and ex vivo structures to patient aggregates remains unclear, particularly for developing therapeutic or diagnostic molecules. Cryo-electron microscopy (cryo-EM) studies of αSyn fibrils with ligands often reveal binding at multiple sites, likely due to high ligand concentrations. Here, various structural and chemical biology techniques were used to characterize αSyn fibrils in the presence of EX-6, a candidate ligand for positron emission tomography (PET) imaging of synucleinopathies. Transmission electron microscopy (TEM) and cryo-EM revealed no significant fibril core changes upon binding. Förster resonance energy transfer (FRET) further demonstrated that the disordered C-terminus was unaltered. Cryo-EM and crosslinking mass spectrometry (XL-MS) identified consistent binding sites, with one (Site 2*) providing a well-defined pocket for high-resolution analysis. Site 2* showed similar residue positioning in MSA patient-derived structures, suggesting MSA selectivity. [ 3 H]-EX-6 binding assays demonstrated a 10-fold preference for MSA over PD tissue, with autoradiography further confirming MSA selectivity. Taken together, the combined use of structural and chemical biology techniques provides a comprehensive understanding of EX-6 binding that would not be possible with any single method. Optimization of ligand-protein and ligand-ligand interactions observed in the cryo-EM structure will enable the development of EX-6 as a PET imaging probe for MSA.

Respiratory syncytial virus (RSV) is an enveloped, filamentous, negative-strand RNA virus that causes significant respiratory illness worldwide. RSV vaccines are available, however there is still significant need for research to support the development of vaccines and therapeutics against RSV and related Mononegavirales viruses. Individual virions vary in size, with an average diameter of ~130 nm and ranging from ~500 nm to over 10 µm in length. Though the general arrangement of structural proteins in virions is known, we use cryo-electron tomography and sub-tomogram averaging to determine the molecular organization of RSV structural proteins. We show that the peripheral membrane-associated RSV matrix (M) protein is arranged in a packed helical-like lattice of M-dimers. We report that RSV F glycoprotein is frequently observed as pairs of trimers oriented in an anti-parallel conformation to support potential interactions between trimers. Our sub-tomogram averages indicate the positioning of F-trimer pairs is correlated with the underlying M lattice. These results provide insight into RSV virion organization and may aid in the development of RSV vaccines and anti-viral targets.

Eosinophils are white blood cells that participate in innate immune responses and have an essential role in the pathogenesis of inflammatory and neoplastic disorders. Upon activation, eosinophils release cytotoxic proteins such as major basic protein-1 (MBP-1) from cytoplasmic secretory granules (SGr) wherein MBP-1 is stored as nanocrystals. How the MBP-1 nanocrystalline core is formed, stabilized, and subsequently mobilized remains unknown. Here, we report the in-situ structure of crystalline MBP-1 within SGrs of human eosinophils. The structure reveals a mechanism for intragranular crystal packing and stabilization of MBP-1 via a structurally conserved loop region that is associated with calcium-dependent carbohydrate binding in other C-type lectin (CTL) proteins. Single-cell and single-SGr profiling correlating real-space three-dimensional information from cellular montage cryo-electron tomography (cryo-ET) and microcrystal electron diffraction (MicroED) data obtained from non-activated and IL33-activated eosinophils revealed activation-dependent crystal expansion and extrusion of expanded crystals from SGr. These results suggest that MBP-1 crystals play a dynamic role in the release of SGr contents. Collectively, this research demonstrates the importance of in-situ macromolecular structure determination.

Correlative light and electron microscopy (CLEM) pipelines serve to integrate the imaging modalities of fluorescence light microscopy (FLM) and cryogenic electron microscopy (cryo-EM) to produce contextually relevant high-resolution structural snapshots of biological systems. Innovations in sample preparation, instrumentation, imaging, and data processing have advanced the field of cryo-EM. This review focuses on prior work and recent developments in the field of cryo- EM that support further integration of technologies for correlative microscopy workflows.

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.

A number of extracellular helical protein polymers are crucial for supporting bacterial motility. The bacterial flagellum is a polymeric appendage used to support cellular motility. Historically, structural studies of flagellar and other filaments were limited to those present as or locked into straightened states. Here, we present a robust workflow that produces biologically relevant high-resolution cryo-electron microscopy (cryo-EM) structures of bacterial flagellar filaments. We highlight how a simple purification method, centered around several centrifugation steps, exploits the process of filament ejection in Caulobacter crescentus and results in isolated filaments amenable to transmission electron microscopy (TEM) studies. The quality of the sample is validated by SDS-PAGE and negative stain TEM analysis before a sample is vitrified for cryogenic electron microscopy (cryo-EM) data collection. We provide a detailed protocol for reconstructing either straight or curved flagellar filaments by cryo-EM helical reconstruction methods, followed by an overview of model building and validation. In our hands, this workflow resulted in several flagellar structures below 3 Å resolution, with one data set reaching a global resolution of 2.1 Å. The application of this workflow supports structure-function studies to better understand the molecular interactions that regulate filament architecture in biologically relevant states. Future work will not only examine interactions that regulate bacterial flagellar and other filament organization but also provide a foundation for developing new helical biopolymers for biotech applications. Key features • Rapid high-quality purification of bacterial flagella via simple bacterial culturing, centrifugation, and resuspension methods. • High-throughput cryo-EM data collection of filamentous objects. • Use of cryoSPARC implementations of helical reconstruction algorithms to generate high-resolution 3D structures of bacterial flagella or other helical polymers.