Cryo-electron microscopy in the study of virus entry and infection.

Viruses have been responsible for many epidemics and pandemics that have impacted human life globally. The COVID-19 pandemic highlighted both our vulnerability to viral outbreaks, as well as the mobilization of the scientific community to come together to combat the unprecedented threat to humanity. Cryo-electron microscopy (cryo-EM) played a central role in our understanding of SARS-CoV-2 during the pandemic and continues to inform about this evolving pathogen. Cryo-EM with its two popular imaging modalities, single particle analysis (SPA) and cryo-electron tomography (cryo-ET), has contributed immensely to understanding the structure of viruses and interactions that define their life cycles and pathogenicity. Here, we review how cryo-EM has informed our understanding of three distinct viruses, of which two - HIV-1 and SARS-CoV-2 infect humans, and the third, bacteriophages, infect bacteria. For HIV-1 and SARS-CoV-2 our focus is on the surface glycoproteins that are responsible for mediating host receptor binding, and host and cell membrane fusion, while for bacteriophages, we review their structure, capsid maturation, attachment to the bacterial cell surface and infection initiation mechanism.

Metal fluorides-multi-functional tools for the study of phosphoryl transfer enzymes, a practical guide.

Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein families across all kingdoms of life. They make up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyze these reactions is essential in characterizing the processes they regulate. Metal fluorides can be used as multifunctional tools to study these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogs. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryo-EM, and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal using soft X-rays.

Structure and assembly of the human IL-12 signaling complex.

Interleukin (IL)-12 is a heterodimeric pro-inflammatory cytokine. Our cryoelectron microscopy structure determination of human IL-12 in complex with IL-12Rß1 and IL-12Rß2 at a resolution of 3.75 Å reveals that IL-12Rß2 primarily interacts with the IL-12p35 subunit via its N-terminal Ig-like domain, while IL-12Rß1 binds to the p40 subunit with its N-terminal fibronectin III domain. This binding mode of IL-12 with its receptors is similar to that of IL-23 but shows notable differences with other cytokines. Through structural information and biochemical assays, we identified Y62, Y189, and K192 as key residues in IL-12p35, which bind to IL-12Rß2 with high affinity and mediate IL-12 signal transduction. Furthermore, structural comparisons reveal two distinctive conformational states and structural plasticity of the heterodimeric interface in IL-12. As a result, our study advances our understanding of IL-12 signal initiation and opens up new opportunities for the engineering and therapeutic targeting of IL-12.

Zinc transporter 1 functions in copper uptake and cuproptosis.

Copper (Cu) is a co-factor for several essential metabolic enzymes. Disruption of Cu homeostasis results in genetic diseases such as Wilson's disease. Here, we show that the zinc transporter 1 (ZnT1), known to export zinc (Zn) out of the cell, also mediates Cu2+ entry into cells and is required for Cu2+-induced cell death, cuproptosis. Structural analysis and functional characterization indicate that Cu2+ and Zn2+ share the same primary binding site, allowing Zn2+ to compete for Cu2+ uptake. Among ZnT members, ZnT1 harbors a unique inter-subunit disulfide bond that stabilizes the outward-open conformations of both protomers to facilitate efficient Cu2+ transport. Specific knockout of the ZnT1 gene in the intestinal epithelium caused the loss of Lgr5+ stem cells due to Cu deficiency. ZnT1, therefore, functions as a dual Zn2+ and Cu2+ transporter and potentially serves as a target for using Zn2+ in the treatment of Wilson's disease caused by Cu overload.

Structural determinants of DNA cleavage by a CRISPR HNH-Cascade system.

Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. Recently, a highly precise subtype I-F1 CRISPR-Cas system (HNH-Cascade) was found that lacks Cas3, the absence of which is compensated for by the insertion of an HNH endonuclease domain in the Cas8 Cascade component. Here, we describe the cryo-EM structure of Selenomonas sp. HNH-Cascade (SsCascade) in complex with target DNA and characterize its mechanism of action. The Cascade scaffold is complemented by the HNH domain, creating a ring-like structure in which the unwound target DNA is precisely cleaved. This structure visualizes a unique hybrid of two extensible biological systems-Cascade, an evolutionary platform for programmable DNA effectors, and an HNH nuclease, an adaptive domain with a spectrum of enzymatic activity.

Cryo-EM structure of monomeric CXCL12-bound CXCR4 in the active state.

CXCR4 binding of its endogenous agonist CXCL12 leads to diverse functions, including bone marrow retention of hematopoietic progenitors and cancer metastasis. However, the structure of the CXCL12-bound CXCR4 remains unresolved despite available structures of CXCR4 in complex with antagonists. Here, we present the cryoelectron microscopy (cryo-EM) structure of the CXCL12-CXCR4-Gi complex at an overall resolution of 2.65 Å. CXCL12 forms a 1:1 stoichiometry complex with CXCR4, following the two-site model. The first 8 amino acids of mature CXCL12 are crucial for CXCR4 activation by forming polar interactions with minor sub-pocket residues in the transmembrane binding pocket. The 3.2-Å distance between V3 of CXCL12 and the "toggle switch" W6.48 marks the deepest insertion among all chemokine-receptor pairs, leading to conformational changes of CXCR4 for G protein activation. These results, combined with functional assays and computational analysis, provide the structural basis for CXCR4 activation by CXCL12.

Structural basis of psychedelic LSD recognition at dopamine D1 receptor.

Understanding the kinetics of LSD in receptors and subsequent induced signaling is crucial for comprehending both the psychoactive and therapeutic effects of LSD. Despite extensive research on LSD's interactions with serotonin 2A and 2B receptors, its behavior on other targets, including dopamine receptors, has remained elusive. Here, we present cryo-EM structures of LSD/PF6142-bound dopamine D1 receptor (DRD1)-legobody complexes, accompanied by a ß-arrestin-mimicking nanobody, NBA3, shedding light on the determinants of G protein coupling versus ß-arrestin coupling. Structural analysis unveils a distinctive binding mode of LSD in DRD1, particularly with the ergoline moiety oriented toward TM4. Kinetic investigations uncover an exceptionally rapid dissociation rate of LSD in DRD1, attributed to the flexibility of extracellular loop 2 (ECL2). Moreover, G protein can stabilize ECL2 conformation, leading to a significant slowdown in ligand's dissociation rate. These findings establish a solid foundation for further exploration of G protein-coupled receptor (GPCR) dynamics and their relevance to signal transduction.

Hybrid immunity to SARS-CoV-2 arises from serological recall of IgG antibodies distinctly imprinted by infection or vaccination.

We describe the molecular-level composition of polyclonal immunoglobulin G (IgG) anti-spike antibodies from ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, vaccination, or their combination ("hybrid immunity") at monoclonal resolution. Infection primarily triggers S2/N-terminal domain (NTD)-reactive antibodies, whereas vaccination mainly induces anti-receptor-binding domain (RBD) antibodies. This imprint persists after secondary exposures wherein >60% of ensuing hybrid immunity derives from the original IgG pool. Monoclonal constituents of the original IgG pool can increase breadth, affinity, and prevalence upon secondary exposures, as exemplified by the plasma antibody SC27. Following a breakthrough infection, vaccine-induced SC27 gained neutralization breadth and potency against SARS-CoV-2 variants and zoonotic viruses (half-maximal inhibitory concentration [IC50] ∼0.1-1.75 nM) and increased its binding affinity to the protective RBD class 1/4 epitope (dissociation constant [KD] < 5 pM). According to polyclonal escape analysis, SC27-like binding patterns are common in SARS-CoV-2 hybrid immunity. Our findings provide a detailed molecular definition of immunological imprinting and show that vaccination can produce class 1/4 (SC27-like) IgG antibodies circulating in the blood.

Purification and Cryo-Electron Microscopy Analysis of Bacterial Appendages.

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.

Protocol for the purification of replisomes from the Xenopus laevis egg extract system for single-particle cryo-EM analysis.

Here, we present a large-scale FLAG immunoprecipitation protocol to isolate large protein complexes driving DNA replication at replicating chromatin assembled in Xenopus laevis egg extract. We describe how to prepare demembranated sperm nuclei (DNA) and low-speed supernatant egg extract (LSS) and present detailed procedures for sample preparation and application onto grids for negative stain electron microscopy (NS-EM) and cryoelectron microscopy (cryo-EM). For complete details on the use and execution of this protocol, please refer to Cvetkovic et al.1.

Reconstitution of the Melibiose Permease of Salmonella enterica serovar Typhimurium (MelBSt) into Lipid Nanodiscs.

Membrane proteins play critical roles in cell physiology and pathology. The conventional way to study membrane proteins at protein levels is to use optimal detergents to extract proteins from membranes. Identification of the optimal detergent is tedious , and in some cases, the protein functions are compromised. While this detergent-based approach has produced meaningful results in membrane protein research, a lipid environment should be more suitable to recapture the protein's native folding and functions. This protocol describes how to prepare amphipathic membrane scaffold-proteins (MSPs)-based nanodiscs of a cation-coupled melibiose symporter of Salmonella enterica serovar Typhimurium (MelBSt), a member of the major facilitator superfamily. MSPs generate nano-assemblies containing membrane proteins surrounded by a patch of native lipids to better preserve their native conformations and functions. This protocol requires purified membrane protein in detergents, purified MSPs in solution, and detergent-destabilized phospholipids. The mixture of all three components at specific ratios is incubated in the presence of Bio-Beads SM-2 resins, which absorb all detergent molecules, allowing the membrane protein to associate with lipids surrounded by the MSPs. By reconstituting the purified membrane proteins back into their native-like lipid environment, these nanodisc-like particles can be directly used in cryo-EM single-particle analysis for structure determination and other biophysical analyses. It is noted that nanodiscs may potentially limit the dynamics of membrane proteins due to suboptimal nanodisc size compared to the native lipid bilayer. Key features • This protocol was built based on the method originally developed by Sligar et al. [1] and modified for a specific major facilitator superfamily transporter • This protocol is robust and reproducible • Lipid nanodiscs can increase membrane protein stability, and reconstituted transporters in lipid nanodiscs can regain function if their function is compromised using detergents • The reconstituted lipids nanodisc can be used for cryo-EM single-particle analysis.

Bayesian reweighting of biomolecular structural ensembles using heterogeneous cryo-EM maps with the cryoENsemble method.

Cryogenic electron microscopy (cryo-EM) has emerged as a powerful method for the determination of structures of complex biological molecules. The accurate characterisation of the dynamics of such systems, however, remains a challenge. To address this problem, we introduce cryoENsemble, a method that applies Bayesian reweighting to conformational ensembles derived from molecular dynamics simulations to improve their agreement with cryo-EM data, thus enabling the extraction of dynamics information. We illustrate the use of cryoENsemble to determine the dynamics of the ribosome-bound state of the co-translational chaperone trigger factor (TF). We also show that cryoENsemble can assist with the interpretation of low-resolution, noisy or unaccounted regions of cryo-EM maps. Notably, we are able to link an unaccounted part of the cryo-EM map to the presence of another protein (methionine aminopeptidase, or MetAP), rather than to the dynamics of TF, and model its TF-bound state. Based on these results, we anticipate that cryoENsemble will find use for challenging heterogeneous cryo-EM maps for biomolecular systems encompassing dynamic components.

Uncoated gold nanoparticles create fewer and less localized defects in model prokaryotic than in model eukaryotic lipid membranes.

The rise of the populations of antibiotic resistant bacteria represents an increasing threat to human health. In addition to the synthesis of new antibiotics, which is an extremely expensive and time-consuming process, one of the ways to combat bacterial infections is the use of gold nanoparticles (Au NPs) as the vehicles for targeted delivery of therapeutic drugs. Since such a strategy requires the investigation of the effect of Au NPs (with and without drugs) on both bacterial and human cells, we investigated how the presence of coating-free Au NPs affects the physicochemical properties of lipid membranes that model prokaryotic (PRO) and eukaryotic (EU) cells. PRO/EU systems prepared as multilamellar liposomes (MLVs) and hybrid structures (HSs) from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG)/1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) in the absence (MLVs)/presence (HSs) of differently distributed Au NPs (sizes ∼20 nm) reported stabilization of the gel phase of PRO systems in comparison with EU one (DSC data of PRO/EU were Tm(MLVs) ≈ 41.8 °C/42.0 °C, Tm¯ (HSs) ≈ 43.1 °C/42.4 °C, whereas UV-Vis response Tm(MLVs) ≈ 41.5 °C/42.0 °C, Tm¯ (HSs) ≈ 42.9 °C/41.1 °C). Vibrational spectroscopic data unraveled a substantial impact of Au NPs on the non-polar part of lipid bilayers, emphasizing the increase of kink and gauche conformers of the hydrocarbon chain. By interpreting the latter as Au NPs-induced defects, which exert the greatest effect when Au NPs are found exclusively outside the lipid membrane, these findings suggested that Au NPs reduced the compactness of EU-based lipid bilayers much more than in analogous PRO systems. Since the uncoated Au NPs manifested adverse effects when applied as antimicrobials, the results obtained in this work contribute towards recognizing AuNP functionalization as a strategy in tuning and reversing this effect.

Structure of biofilm-forming functional amyloid PSMα1 from Staphylococcus aureus.

Biofilm-protected pathogenic Staphylococcus aureus causes chronic infections that are difficult to treat. An essential building block of these biofilms are functional amyloid fibrils that assemble from phenol-soluble modulins (PSMs). PSMα1 cross-seeds other PSMs into cross-ß amyloid folds and is therefore a key element in initiating biofilm formation. However, the paucity of high-resolution structures hinders efforts to prevent amyloid assembly and biofilm formation. Here, we present a 3.5 Å resolution density map of the major PSMα1 fibril form revealing a left-handed cross-ß fibril composed of two C2-symmetric U-shaped protofilaments whose subunits are unusually tilted out-of-plane. Monomeric α-helical PSMα1 is extremely cytotoxic to cells, despite the moderate toxicity of the cross-ß fibril. We suggest mechanistic insights into the PSM functional amyloid formation and conformation transformation on the path from monomer-to-fibril formation. Details of PSMα1 assembly and fibril polymorphism suggest how S. aureus utilizes functional amyloids to form biofilms and establish a framework for developing therapeutics against infection and antimicrobial resistance.

Structural basis for the transport and substrate selection of human urate transporter 1.

High serum urate levels are the major risk factor for gout. URAT1, the primary transporter for urate absorption in the kidneys, is well known as an anti-hyperuricemia drug target. However, the clinical application of URAT1-targeted drugs is limited because of their low specificity and severe side effects. The lack of structural information impedes elucidation of the transport mechanism and the development of new drugs. Here, we present the cryoelectron microscopy (cryo-EM) structures of human URAT1(R477S), its complex with urate, and its closely related homolog OAT4. URAT1(R477S) and OAT4 exhibit major facilitator superfamily (MFS) folds with outward- and inward-open conformations, respectively. Structural comparison reveals a 30° rotation between the N-terminal and C-terminal domains, supporting an alternating access mechanism. A conserved arginine (OAT4-Arg473/URAT1-Arg477) is found to be essential for chloride-mediated inhibition. The URAT1(R477S)-urate complex reveals the specificity of urate recognition. Taken together, our study promotes our understanding of the transport mechanism and substrate selection of URAT1.

Cryo-EM structures of cotton wool plaques' amyloid ß and of tau filaments in dominantly inherited Alzheimer disease.

Cotton wool plaques (CWPs) have been described as features of the neuropathologic phenotype of dominantly inherited Alzheimer disease (DIAD) caused by some missense and deletion mutations in the presenilin 1 (PSEN1) gene. CWPs are round, eosinophilic amyloid-ß (Aß) plaques that lack an amyloid core and are recognizable, but not fluorescent, in Thioflavin S (ThS) preparations. Amino-terminally truncated and post-translationally modified Aß peptide species are the main component of CWPs. Tau immunopositive neurites may be present in CWPs. In addition, neurofibrillary tangles coexist with CWPs. Herein, we report the structure of Aß and tau filaments isolated from brain tissue of individuals affected by DIAD caused by the PSEN1 V261I and A431E mutations, with the CWP neuropathologic phenotype. CWPs are predominantly composed of type I Aß filaments present in two novel arrangements, type Ic and type Id; additionally, CWPs contain type I and type Ib Aß filaments. Tau filaments have the AD fold, which has been previously reported in sporadic AD and DIAD. The formation of type Ic and type Id Aß filaments may be the basis for the phenotype of CWPs. Our data are relevant for the development of PET imaging methodologies to best detect CWPs in DIAD.

Cryo-EM reconstruction of oleate hydratase bound to a phospholipid membrane bilayer.

Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyA•membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20-30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme.

Structures of Mature and Urea-Treated Empty Bacteriophage T5: Insights into Siphophage Infection and DNA Ejection.

T5 is a siphophage that has been extensively studied by structural and biochemical methods. However, the complete in situ structures of T5 before and after DNA ejection remain unknown. In this study, we used cryo-electron microscopy (cryo-EM) to determine the structures of mature T5 (a laboratory-adapted, fiberless T5 mutant) and urea-treated empty T5 (lacking the tip complex) at near-atomic resolutions. Atomic models of the head, connector complex, tail tube, and tail tip were built for mature T5, and atomic models of the connector complex, comprising the portal protein pb7, adaptor protein p144, and tail terminator protein p142, were built for urea-treated empty T5. Our findings revealed that the aforementioned proteins did not undergo global conformational changes before and after DNA ejection, indicating that these structural features were conserved among most myophages and siphophages. The present study elucidates the underlying mechanisms of siphophage infection and DNA ejection.

CheckMyMetal (CMM): validating metal-binding sites in X-ray and cryo-EM data.

Identifying and characterizing metal-binding sites (MBS) within macromolecular structures is imperative for elucidating their biological functions. CheckMyMetal (CMM) is a web based tool that facilitates the interactive validation of MBS in structures determined through X-ray crystallography and cryo-electron microscopy (cryo-EM). Recent updates to CMM have significantly enhanced its capability to efficiently handle large datasets generated from cryo-EM structural analyses. In this study, we address various challenges inherent in validating MBS within both X-ray and cryo-EM structures. Specifically, we examine the difficulties associated with accurately identifying metals and modeling their coordination environments by considering the ongoing reproducibility challenges in structural biology and the critical importance of well annotated, high-quality experimental data. CMM employs a sophisticated framework of rules rooted in the valence bond theory for MBS validation. We explore how CMM validation parameters correlate with the resolution of experimentally derived structures of macromolecules and their complexes. Additionally, we showcase the practical utility of CMM by analyzing a representative cryo-EM structure. Through a comprehensive examination of experimental data, we demonstrate the capability of CMM to advance MBS characterization and identify potential instances of metal misassignment.

Structure and replication of Pseudomonas aeruginosa phage JBD30.

Bacteriophages are the most abundant biological entities on Earth, but our understanding of many aspects of their lifecycles is still incomplete. Here, we have structurally analysed the infection cycle of the siphophage Casadabanvirus JBD30. Using its baseplate, JBD30 attaches to Pseudomonas aeruginosa via the bacterial type IV pilus, whose subsequent retraction brings the phage to the bacterial cell surface. Cryo-electron microscopy structures of the baseplate-pilus complex show that the tripod of baseplate receptor-binding proteins attaches to the outer bacterial membrane. The tripod and baseplate then open to release three copies of the tape-measure protein, an event that is followed by DNA ejection. JBD30 major capsid proteins assemble into procapsids, which expand by 7% in diameter upon filling with phage dsDNA. The DNA-filled heads are finally joined with 180-nm-long tails, which bend easily because flexible loops mediate contacts between the successive discs of major tail proteins. It is likely that the structural features and replication mechanisms described here are conserved among siphophages that utilize the type IV pili for initial cell attachment.