Cryo-Electron Microscopy in the Study of Antiviral Innate Immunity.

Cryo-electron microscopy is a powerful methodology in structural biology and has been broadly used in high-resolution structure determination for challenging samples, which are not readily available for traditional techniques. In particular, the strength of super macro-complexes and the lack of a need for crystals for cryo-EM make this technique feasible for the structural study of complexes involved in antiviral innate immunity. This chapter presents detailed information and experimental procedures of Cryo-EM for determining the structures of the complexes using STING as an example. The procedures included a sample quality check, high-resolution data acquisition, and image processing for Cryo-EM 3D structure determination.

Ejectosome of Pectobacterium bacteriophage ΦM1.

Podophages that infect gram-negative bacteria, such as Pectobacterium pathogen ΦM1, encode tail assemblies too short to extend across the complex gram-negative cell wall. To overcome this, podophages encode a large protein complex (ejectosome) packaged inside the viral capsid and correspondingly ejected during infection to form a transient channel that spans the periplasmic space. Here, we describe the ejectosome of bacteriophage ΦM1 to a resolution of 3.32 Å by single-particle cryo-electron microscopy (cryo-EM). The core consists of tetrameric and octameric ejection proteins which form a ∼1.5-MDa ejectosome that must transition through the ∼30 Å aperture created by the short tail nozzle assembly that acts as the conduit for the passage of DNA during infection. The ejectosome forms several grooves into which coils of genomic DNA are fit before the DNA sharply turns and goes down the tunnel and into the portal. In addition, we reconstructed the icosahedral capsid and hybrid tail apparatus to resolutions between 3.04 and 3.23 Å, and note an uncommon fold adopted by the dimerized decoration proteins which further emphasize the structural diversity of podophages. These reconstructions have allowed the generation of a complete atomic model of the ΦM1, uncovering two distinct decoration proteins and highlighting the exquisite structural diversity of tailed bacteriophages.

EMhub: a web platform for data management and on-the-fly processing in scientific facilities.

Most scientific facilities produce large amounts of heterogeneous data at a rapid pace. Managing users, instruments, reports and invoices presents additional challenges. To address these challenges, EMhub, a web platform designed to support the daily operations and record-keeping of a scientific facility, has been introduced. EMhub enables the easy management of user information, instruments, bookings and projects. The application was initially developed to meet the needs of a cryoEM facility, but its functionality and adaptability have proven to be broad enough to be extended to other data-generating centers. The expansion of EMHub is enabled by the modular nature of its core functionalities. The application allows external processes to be connected via a REST API, automating tasks such as folder creation, user and password generation, and the execution of real-time data-processing pipelines. EMhub has been used for several years at the Swedish National CryoEM Facility and has been installed in the CryoEM center at the Structural Biology Department at St. Jude Children's Research Hospital. A fully automated single-particle pipeline has been implemented for on-the-fly data processing and analysis. At St. Jude, the X-Ray Crystallography Center and the Single-Molecule Imaging Center have already expanded the platform to support their operational and data-management workflows.

Engineering substrate channeling in a bifunctional terpene synthase.

Fusicoccadiene synthase from Phomopsis amygdala (PaFS) is a bifunctional terpene synthase. It contains a prenyltransferase (PT) domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate, and a cyclase domain that converts GGPP into fusicoccadiene, a precursor of the diterpene glycoside Fusicoccin A. The two catalytic domains are connected by a flexible 69-residue linker. The PT domain mediates oligomerization to form predominantly octamers, with cyclase domains randomly splayed out around the PT core. Surprisingly, despite the random positioning of cyclase domains, substrate channeling is operative in catalysis since most of the GGPP generated by the PT remains on the enzyme for cyclization. Here, we demonstrate that covalent linkage of the PT and cyclase domains is not required for GGPP channeling, although covalent linkage may improve channeling efficiency. Moreover, GGPP competition experiments with other diterpene cyclases indicate that the PaFS PT and cyclase domains are preferential partners regardless of whether they are covalently linked or not. The cryoelectron microscopy structure of the 600-kD "linkerless" construct, in which the 69-residue linker is spliced out and replaced with the tripeptide PTQ, reveals that cyclase pairs associate with all four sides of the PT octamer and exhibit fascinating quaternary structural flexibility. These results suggest that optimal substrate channeling is achieved when a cyclase domain associates with the side of the PT octamer, regardless of whether the two domains are covalently linked and regardless of whether this interaction is transient or locked in place.

A binding site for the antibiotic GE81112 in the ribosomal mRNA channel.

The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B and B1 with molecular masses of 643-658 Da). Previous biochemical and structural characterisation of GE81112 indicates that the primary mechanism of action of this antibiotic is to (1) prevent the initiator tRNA from binding correctly to the P-site and (2) block conformational rearrangements in initiation factor IF3, resulting in an unlocked 30S pre/C state. In this study, using cryoEM, we have determined the binding site of GE81112 in initiation complexes (3.2-3.7Å) and on empty ribosomes (2.09 Å). This binding site is within the mRNA channel (E-site) but remote from the binding site of the initiation factors and initiator tRNA. This suggests that it acts allosterically to prevent the initiator tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.

Rapid small-scale nanobody-assisted purification of ryanodine receptors for cryo-EM.

Ryanodine receptors (RyRs) are large Ca2+ release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of which has been associated with a series of life-threatening diseases. The need for large amounts of native tissue or eukaryotic cell cultures limits advances in structural studies of RyRs. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification process, from isolated membranes to cryo-EM grade protein, is achieved within 4 h on the bench, yielding protein usable for cryo-EM analysis. This is demonstrated by solving the structures of rabbit RyR1, solubilized in detergent, reconstituted into lipid nanodiscs or liposomes, and bovine RyR2 reconstituted in nanodisc, and mouse RyR2 in detergent. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in cases where the amount of starting material is limited.

Real-space heterogeneous reconstruction, refinement, and disentanglement of CryoEM conformational states with HetSIREN.

Single-particle analysis by Cryo-electron microscopy (CryoEM) provides direct access to the conformation of each macromolecule. However, the image's signal-to-noise ratio is low, and some form of classification is usually performed at the image processing level to allow structural modeling. Classical classification methods imply the existence of a discrete number of structural conformations. However, new heterogeneity algorithms introduce a novel reconstruction paradigm, where every state is represented by a lower number of particles, potentially just one, allowing the estimation of conformational landscapes representing the different structural states a biomolecule explores. In this work, we present a novel deep learning-based method called HetSIREN. HetSIREN can fully reconstruct or refine a CryoEM volume in real space based on the structural information summarized in a conformational latent space. The unique characteristics that set HetSIREN apart start with the definition of the approach as a real space-based only method, a fact that allows spatially focused analysis, but also the introduction of a novel network architecture specifically designed to make use of meta-sinusoidal activations, with proven high analytics capacities. Continuing with innovations, HetSIREN can also refine the pose parameters of the images at the same time that it conditions the network with prior information/constraints on the maps, such as Total Variation and L 1 denoising, ultimately yielding cleaner volumes with high-quality structural features. Finally, but very importantly, HetSIREN addresses one of the most confusing issues in heterogeneity analysis, as it is the fact that real structural heterogeneity estimation is entangled with pose estimation (and to a lesser extent with CTF estimation), in this way, HetSIREN introduces a novel encoding architecture able to decouple pose and CTF information from the conformational landscape, resulting in more accurate and interpretable conformational latent spaces. We present results on computer-simulated data, public data from EMPIAR, and data from experimental systems currently being studied in our laboratories. An important finding is the sensitivity of the structure and dynamics of the SARS-CoV-2 Spike protein on the storage temperature.

Structural Basis for Monoclonal Antibody Therapy for Transthyretin Amyloidosis.

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.

Crossing the membrane-What does it take to flip a phospholipid? Structural and biochemical advances on P4-ATPase flippases.

Membrane asymmetry is critical for maintenance of several different processes such as cell signaling, apoptosis, and vesicular transport in various eukaryotic systems. Flippases of the P4-ATPase family are associated with flipping phospholipids from the luminal or exoplasmic leaflet to the cytosolic leaflet. P4-ATPases belong to the P-type ATPase family, which are activated by phosphorylation and couple ATPase activity to substrate translocation. These proteins possess a transmembrane domain responsible for substrate transport, while the cytosolic machinery performs the necessary ATP hydrolysis for this process. Several high-resolution structures of human or yeast P4-ATPases have recently been resolved, but a comprehensive overview of the changes for reaction cycle in different members was crucial for future research. In this review, we have compiled available data reflecting the reaction cycle-associated changes in conformation of P4-ATPases. Together, this will provide an improved understanding of the similarities and differences between these members, which will drive further structural, functional, and computational studies to understand the mechanisms of these flippases.

Established and emerging players in phospholipid scrambling: A structural perspective.

The maintenance of a diverse and non-homogeneous lipid composition in cell membranes is crucial for a multitude of cellular processes. One important example is transbilayer lipid asymmetry, which refers to a difference in lipid composition between the two leaflets of a cellular membrane. Transbilayer asymmetry is especially pronounced at the plasma membrane, where at resting state, negatively-charged phospholipids such as phosphatidylserine (PS) are almost exclusively restricted to the cytosolic leaflet, whereas sphingolipids are mostly found in the exoplasmic leaflet. Transbilayer movement of lipids is inherently slow, and for a fast cellular response, for example during apoptosis, transmembrane proteins termed scramblases facilitate the movement of polar/charged lipid headgroups through the membrane interior. In recent years, an expanding number of proteins from diverse families have been suggested to possess a lipid scramblase activity. Members of TMEM16 and XKR proteins have been implicated in blood clotting and apoptosis, whereas the scrambling activity of ATG9 and TMEM41B/VMP1 proteins contributes to the synthesis of autophagosomal membrane during autophagy. Structural studies, in vitro reconstitution of lipid scrambling, and molecular dynamics simulations have significantly advanced our understanding of the molecular mechanisms of lipid scrambling and helped delineate potential lipid transport pathways through the membrane. A number of examples also suggest that lipid scrambling activity can be combined with another activity, as is the case for TMEM16 proteins, which also function as ion channels, rhodopsin in the photoreceptor membrane, and possibly other G-protein coupled receptors.

Sec18 side-loading is essential for universal SNARE recycling across cellular contexts.

SNARE proteins drive membrane fusion as their core domains zipper into a parallel four-helix bundle1,2. After fusion, these bundles are disassembled by the AAA+ protein Sec18/NSF and its adaptor Sec17/ α-SNAP3,4 to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains5. Previous structures of the NSF-α-SNAP-SNARE complex revealed SNARE domain threaded through the D1 ATPase ring6, posing a topological constraint as SNARE transmembrane domains would prevent complete substrate threading as suggested for other AAA+ systems7. Here, in vivo mass-spectrometry reveals N-terminal SNARE domain interactions with Sec18, exacerbating this topological issue. Cryo-EM structures of a yeast SNARE complex, Sec18, and Sec17 in a non-hydrolyzing condition shows SNARE Sso1 threaded through the D1 and D2 ATPase rings of Sec18, with its folded, N-terminal Habc domain interacting with the D2 ring. This domain does not unfold during Sec18/NSF activity. Cryo-EM structures under hydrolyzing conditions revealed substrate-released and substrate-free states of Sec18 with a coordinated opening in the side of the ATPase rings. Thus, Sec18/NSF operates by substrate side-loading and unloading topologically constrained SNARE substrates.

Architecture of CTPS filament networks revealed by cryo-electron tomography.

The cytoophidium is a novel type of membraneless organelle, first observed in the ovaries of Drosophila using fluorescence microscopy. In vitro, purified Drosophila melanogaster CTPS (dmCTPS) can form metabolic filaments under the presence of either substrates or products, and their structures that have been analyzed using cryo-electron microscopy (cryo-EM). These dmCTPS filaments are considered the fundamental units of cytoophidia. However, due to the resolution gap between light and electron microscopy, the precise assembly pattern of cytoophidia remains unclear. In this study, we find that dmCTPS filaments can spontaneously assemble in vitro, forming network structures that reach micron-scale dimensions. Using cryo-electron tomography (cryo-ET), we reconstruct the network structures formed by dmCTPS filaments under substrate or product binding conditions and elucidate their assembly process. The dmCTPS filaments initially form structural bundles, which then further assemble into larger networks. By identifying, tracking, and statistically analyzing the filaments, we observed distinct characteristics of the structural bundles formed under different conditions. This study provides the first systematic analysis of dmCTPS filament networks, offering new insights into the relationship between cytoophidia and metabolic filaments.

Algebraic constraints and algorithms for common lines in cryo-EM.

We revisit the topic of common lines between projection images in single-particle cryo-electron microscopy (cryo-EM). We derive a novel low-rank constraint on a certain 2n × n matrix storing properly scaled basis vectors for the common lines between n projection images of one molecular conformation. Using this algebraic constraint and others, we give optimization algorithms to denoise common lines and recover the unknown 3D rotations associated with the images. As an application, we develop a clustering algorithm to partition a set of noisy images into homogeneous communities using common lines, in the case of discrete heterogeneity in cryo-EM. We demonstrate the methods on synthetic and experimental datasets.

Cryo-EM structure of ACE2-SIT1 in complex with tiagabine.

The pharmacology of amino acid transporters in the SLC6 family is poorly developed compared to that of the neurotransmitter transporters. To identify new inhibitors of the proline transporter SIT1 (SLC6A20), its expression in Xenopus laevis oocytes was optimized. Trafficking of SIT1 was augmented by co-expression of angiotensin-converting enzyme 2 (ACE2) in oocytes but there was no strict requirement for co-expression of ACE2. A pharmacophore-guided screen identified tiagabine as a potent non-competitive inhibitor of SIT1. To understand its binding mode, we determined the cryo-electron microscopy (cryo-EM) structure of ACE2-SIT1 bound with tiagabine. The inhibitor binds close to the orthosteric proline binding site, but due to its size extends into the cytosolic vestibule. This causes the transporter to adopt an inward-open conformation, in which the intracellular gate is blocked. This study provides the first structural insight into inhibition of SIT1 and generates tools for a better understanding of the ACE2-SIT1 complex. These findings may have significance for SARS-CoV-2 binding to its receptor ACE2 in human lung alveolar cells where SIT1 and ACE2 are functionally expressed.

Structural Basis of Bifunctional CTP/dCTP Synthase.

The final step in the de novo synthesis of cytidine 5'-triphosphate (CTP) is catalyzed by CTP synthase (CTPS), which can form cytoophidia in all three domains of life. Recently, we have discovered that CTPS binds to ribonucleotides (NTPs) to form filaments, and have successfully resolved the structures of Drosophila melanogaster CTPS bound with NTPs. Previous biochemical studies have shown that CTPS can bind to deoxyribonucleotides (dNTPs) to produce 2'-deoxycytidine-5'-triphosphate (dCTP). However, the structural basis of CTPS binding to dNTPs is still unclear. In this study, we find that Drosophila CTPS can also form filaments with dNTPs. Using cryo-electron microscopy, we are able to resolve the structure of Drosophila melanogaster CTPS bound to dNTPs with a resolution of up to 2.7 Å. By combining these structural findings with biochemical analysis, we compare the binding and reaction characteristics of NTPs and dNTPs with CTPS. Our results indicate that the same enzyme can act bifunctionally as CTP/dCTP synthase in vitro, and provide a structural basis for these activities.

Structural analysis of the human C5a-C5aR1 complex using cryo-electron microscopy.

The complement system is a complex network of proteins that plays a crucial role in the innate immune response. One important component of this system is the C5a-C5aR1 complex, which is critical in the recruitment and activation of immune cells. In-depth investigation of the activation mechanism as well as biased signaling of the C5a-C5aR1 system will facilitate the elucidation of C5a-mediated pathophysiology. In this study, we determined the structure of C5a-C5aR1-Gi complex at a high resolution of 3 Å using cryo-electron microscopy (Cryo-EM). Our results revealed the binding site of C5a, which consists of a polar recognition region on the extracellular side and an amphipathic pocket within the transmembrane domain. Furthermore, we found that C5a binding induces conformational changes of C5aR1, which subsequently leads to the activation of G protein signaling pathways. Notably, a key residue (M265) located on transmembrane helix 6 (TM6) was identified to play a crucial role in regulating the recruitment of ß-arrestin driven by C5a. This study provides more information about the structure and function of the human C5a-C5aR1 complex, which is essential for the proper functioning of the complement system. The findings of this study can also provide a foundation for the design of new pharmaceuticals targeting this receptor with bias or specificity.

Recent technical advances in cellular cryo-electron tomography.

Understanding the in situ structure, organization, and interactions of macromolecules is essential for elucidating their functions and mechanisms of action. Cellular cryo-electron tomography (cryo-ET) is a cutting-edge technique that reveals in situ molecular-resolution architectures of macromolecules in their lifelike states. It also provides insights into the three-dimensional distribution of macromolecules and their spatial relationships with various subcellular structures. Thus, cellular cryo-ET bridges the gap between structural biology and cell biology. With rapid advancements, this technique achieved substantial improvements in throughput, automation, and resolution. This review presents the fundamental principles and methodologies of cellular cryo-ET, highlighting recent developments in sample preparation, data collection, and image processing. We also discuss emerging trends and potential future directions. As cellular cryo-ET continues to develop, it is set to play an increasingly vital role in structural cell biology.

Myosin forces elicit an F-actin structural landscape that mediates mechanosensitive protein recognition.

Cells mechanically interface with their surroundings through cytoskeleton-linked adhesions, allowing them to sense physical cues that instruct development and drive diseases such as cancer. Contractile forces generated by myosin motor proteins mediate these mechanical signal transduction processes through unclear protein structural mechanisms. Here, we show that myosin forces elicit structural changes in actin filaments (F-actin) that modulate binding by the mechanosensitive adhesion protein α-catenin. Using correlative cryo-fluorescence microscopy and cryo-electron tomography, we identify F-actin featuring domains of nanoscale oscillating curvature at cytoskeleton-adhesion interfaces enriched in zyxin, a marker of actin-myosin generated traction forces. We next introduce a reconstitution system for visualizing F-actin in the presence of myosin forces with cryo-electron microscopy, which reveals morphologically similar superhelical F-actin spirals. In simulations, transient forces mimicking tugging and release of filaments by motors produce spirals, supporting a mechanistic link to myosin's ATPase mechanochemical cycle. Three-dimensional reconstruction of spirals uncovers extensive asymmetric remodeling of F-actin's helical lattice. This is recognized by α-catenin, which cooperatively binds along individual strands, preferentially engaging interfaces featuring extended inter-subunit distances while simultaneously suppressing rotational deviations to regularize the lattice. Collectively, we find that myosin forces can deform F-actin, generating a conformational landscape that is detected and reciprocally modulated by a mechanosensitive protein, providing a direct structural glimpse at active force transduction through the cytoskeleton.

Cryo-EM structures of a bathy phytochrome histidine kinase reveal a unique light-dependent activation mechanism.

Phytochromes are photoreceptor proteins in plants, fungi, and bacteria. They can adopt two photochromic states with differential biochemical responses. The structural changes transducing the signal from the chromophore to the biochemical output modules are poorly understood due to challenges in capturing structures of the dynamic, full-length protein. Here, we present cryoelectron microscopy (cryo-EM) structures of the phytochrome from Pseudomonas aeruginosa (PaBphP) in its resting (Pfr) and photoactivated (Pr) state. The kinase-active Pr state has an asymmetric, dimeric structure, whereas the kinase-inactive Pfr state opens up. This behavior is different from other known phytochromes and we explain it with the unusually short connection between the photosensory and output modules. Multiple sequence alignment of this region suggests evolutionary optimization for different modes of signal transduction in sensor proteins. The results establish a new mechanism for light-sensing by phytochrome histidine kinases and provide input for the design of optogenetic phytochrome variants.

Structural perspectives on the androgen receptor, the elusive shape-shifter.

The androgen receptor (AR) is a type I nuclear receptor and master transcription factor responsible for development and maintenance of male secondary sex characteristics. Aberrant AR activity is associated with numerous diseases, including prostate cancer, androgen insensitivity syndrome, spinal and bulbar muscular atrophy, and androgenic alopecia. Recent studies have shown that AR adopts numerous conformations that can modulate its ability to bind and transcribe its target DNA substrates, a feature that can be hijacked in the context of cancer. Here, we summarize a series of structural observations describing how this elusive shape-shifter binds to multiple partners, including self-interactions, DNA, and steroid and non-steroidal ligands. We present evidence that AR's pervasive structural plasticity confers an ability to broadly bind and transcribe numerous ligands in the normal and disease state, and explain the structural basis for adaptive resistance mutations to antiandrogen treatment. These evolutionary features are integral to receptor function, and are commonly lost in androgen insensitivity syndrome, or reinforced in cancer.