ABSTRACT
In many viruses, including rotavirus (RV), the major pathogen of infantile gastroenteritis, capping of viral messenger RNAs is a pivotal step for efficient translation of the viral genome. In RV, VP3 caps the nascent transcripts synthesized from the genomic dsRNA segments by the RV polymerase VP1 within the particle core. Here, from cryo-electron microscopy, x-ray crystallography, and biochemical analyses, we show that VP3 forms a stable tetrameric assembly with each subunit having a modular domain organization, which uniquely integrates five distinct enzymatic steps required for capping the transcripts. In addition to the previously known guanylyl- and methyltransferase activities, we show that VP3 exhibits hitherto unsuspected RNA triphosphatase activity necessary for initiating transcript capping and RNA helicase activity likely required for separating the RNA duplex formed transiently during endogenous transcription. From our studies, we propose a new mechanism for how VP3 inside the virion core caps the nascent transcripts exiting from the polymerase.
Subject(s)
Rotavirus , Capsid Proteins/metabolism , Cryoelectron Microscopy , Genome, Viral , Nucleotidyltransferases/metabolism , RNA, Viral/genetics , Rotavirus/genetics , Rotavirus/metabolism , Virion/metabolismABSTRACT
[This corrects the article DOI: 10.1371/journal.pbio.3000311.].
ABSTRACT
SGIV, or Singapore grouper iridovirus, is a large double-stranded DNA virus, reaching a diameter of 220 nm and packaging a genome of 140 kb. We present a 3D cryoelectron microscopy (cryo-EM) icosahedral reconstruction of SGIV determined at 8.6-Å resolution. It reveals several layers including a T = 247 icosahedral outer coat, anchor proteins, a lipid bilayer, and the encapsidated DNA. A new segmentation tool, iSeg, was applied to extract these layers from the reconstructed map. The outer coat was further segmented into major and minor capsid proteins. None of the proteins extracted by segmentation have known atomic structures. We generated models for the major coat protein using three comparative modeling tools, and evaluated each model using the cryo-EM map. Our analysis reveals a new architecture in the Iridoviridae family of viruses. It shares similarities with others in the same family, e.g., Chilo iridescent virus, but also shows new features of the major and minor capsid proteins.
Subject(s)
Capsid Proteins/chemistry , Iridovirus/metabolism , Capsid Proteins/metabolism , Cryoelectron Microscopy , DNA, Viral/chemistry , Iridovirus/chemistry , Iridovirus/genetics , Lipid Bilayers/metabolism , Models, Molecular , Protein ConformationABSTRACT
Single-stranded RNA bacteriophages (ssRNA phages) infect Gram-negative bacteria via a single maturation protein (Mat), which attaches to a retractile pilus of the host. Here we present structures of the ssRNA phage MS2 in complex with the Escherichia coli F-pilus, showing a network of hydrophobic and electrostatic interactions at the Mat-pilus interface. Moreover, binding of the pilus induces slight orientational variations of the Mat relative to the rest of the phage capsid, priming the Mat-connected genomic RNA (gRNA) for its release from the virions. The exposed tip of the attached Mat points opposite to the direction of the pilus retraction, which may facilitate the translocation of the gRNA from the capsid into the host cytosol. In addition, our structures determine the orientation of the assembled F-pilin subunits relative to the cell envelope, providing insights into the F-like type IV secretion systems.
Subject(s)
Escherichia coli/virology , Levivirus/ultrastructure , Cell Wall/metabolism , Cell Wall/ultrastructure , Cell Wall/virology , Cryoelectron Microscopy , Escherichia coli/ultrastructure , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/ultrastructure , Fimbriae Proteins/metabolism , Fimbriae Proteins/ultrastructure , Fimbriae, Bacterial/metabolism , Fimbriae, Bacterial/ultrastructure , Fimbriae, Bacterial/virology , Levivirus/genetics , RNA, Guide, Kinetoplastida/metabolism , RNA, Viral/metabolism , Viral Proteins/ultrastructureABSTRACT
Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted exotoxins-toxin A (TcdA) and toxin B (TcdB)-that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4-1.4E and U3-bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and "kisses" the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the "native" conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti-C. difficile toxin therapeutics.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/physiology , Bacterial Toxins/antagonists & inhibitors , Clostridium Infections/metabolism , Animals , Ankyrin Repeat/genetics , Antibodies, Monoclonal , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Broadly Neutralizing Antibodies , Caco-2 Cells , Clostridioides difficile/metabolism , Clostridioides difficile/pathogenicity , Cryoelectron Microscopy , Enterotoxins/metabolism , Humans , Mice , Protein Engineering/methodsABSTRACT
Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Carrier Proteins/physiology , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/physiology , Escherichia coli/physiology , Lipoproteins/metabolism , Membrane Transport Proteins/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Anti-Bacterial Agents/pharmacology , Carrier Proteins/drug effects , Carrier Proteins/ultrastructure , Cryoelectron Microscopy/methods , Drug Resistance, Multiple, Bacterial/drug effects , Electron Microscope Tomography/methods , Escherichia coli/drug effects , Escherichia coli/ultrastructure , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/drug effects , Escherichia coli Proteins/ultrastructure , Intravital Microscopy/methods , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Peptidoglycan/metabolism , Periplasm/metabolism , Protein Binding/drug effects , Protein Structure, Quaternary/drug effectsABSTRACT
Ribosomes from Mycobacterium tuberculosis (Mtb) possess species-specific ribosomal RNA (rRNA) expansion segments and ribosomal proteins (rProtein). Here, we present the near-atomic structures of the Mtb 50S ribosomal subunit and the complete Mtb 70S ribosome, solved by cryo-electron microscopy. Upon joining of the large and small ribosomal subunits, a 100-nt long expansion segment of the Mtb 23S rRNA, named H54a or the 'handle', switches interactions from with rRNA helix H68 and rProtein uL2 to with rProtein bS6, forming a new intersubunit bridge 'B9'. In Mtb 70S, bridge B9 is mostly maintained, leading to correlated motions among the handle, the L1 stalk and the small subunit in the rotated and non-rotated states. Two new protein densities were discovered near the decoding center and the peptidyl transferase center, respectively. These results provide a structural basis for studying translation in Mtb as well as developing new tuberculosis drugs.
Subject(s)
Mycobacterium tuberculosis/chemistry , Ribosomes/chemistry , Cryoelectron Microscopy , Models, Molecular , Motion , Mycobacterium smegmatis/chemistry , Protein Synthesis Inhibitors , Ribosomal Proteins/chemistry , Ribosome Subunits, Large, Bacterial/chemistry , Species SpecificityABSTRACT
In single-stranded RNA bacteriophages (ssRNA phages) a single copy of the maturation protein binds the genomic RNA (gRNA) and is required for attachment of the phage to the host pilus. For the canonical Allolevivirus Qß the maturation protein, A2, has an additional role as the lysis protein, by its ability to bind and inhibit MurA, which is involved in peptidoglycan biosynthesis. Here, we determined structures of Qß virions, virus-like particles, and the Qß-MurA complex using single-particle cryoelectron microscopy, at 4.7-Å, 3.3-Å, and 6.1-Å resolutions, respectively. We identified the outer surface of the ß-region in A2 as the MurA-binding interface. Moreover, the pattern of MurA mutations that block Qß lysis and the conformational changes of MurA that facilitate A2 binding were found to be due to the intimate fit between A2 and the region encompassing the closed catalytic cleft of substrate-liganded MurA. Additionally, by comparing the Qß virion with Qß virus-like particles that lack a maturation protein, we observed a structural rearrangement in the capsid coat proteins that is required to package the viral gRNA in its dominant conformation. Unexpectedly, we found a coat protein dimer sequestered in the interior of the virion. This coat protein dimer binds to the gRNA and interacts with the buried α-region of A2, suggesting that it is sequestered during the early stage of capsid formation to promote the gRNA condensation required for genome packaging. These internalized coat proteins are the most asymmetrically arranged major capsid proteins yet observed in virus structures.
Subject(s)
Alkyl and Aryl Transferases/metabolism , Allolevivirus/ultrastructure , Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/genetics , Capsid/chemistry , Capsid Proteins/chemistry , Gene Expression Regulation, Viral , Protein Conformation , RNA, Viral , Virion/metabolismABSTRACT
Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-Å cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10-12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.
Subject(s)
Chaperonin 60/chemistry , Cryoelectron Microscopy/methods , Models, Molecular , Chaperonin 60/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Protein Conformation , Protein Domains , Protein Subunits/chemistry , Protein Subunits/metabolismABSTRACT
Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo-EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly. With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.
Subject(s)
Cryoelectron Microscopy , Macromolecular Substances/chemistry , Models, Molecular , Molecular Conformation , Bacteriophage P22 , Binding Sites , Capsid Proteins/chemistry , Cryoelectron Microscopy/methods , Protein Binding , Protein Conformation , Reproducibility of ResultsABSTRACT
Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only the structures of the capsids and their associated protein components have been resolved to high resolution. Qß, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature virion, the maturation protein, A2, binds to the gRNA and is required for adsorption to the F-pilus. Here, we report the cryo-electron microscopy (cryo-EM) structures of Qß with and without symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals A2 and the gRNA. A2 contains a bundle of α-helices and replaces one dimer of coat proteins at a twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity, which asymmetrically expands the surrounding coat protein shell to potentially facilitate RNA release during infection. We observe a fixed pattern of gRNA organization among all viral particles, with the major and minor grooves of RNA helices clearly visible. A single layer of RNA directly contacts every copy of the coat protein, with one-third of the interactions occurring at operator-like RNA hairpins. These RNA-coat interactions stabilize the tertiary structure of gRNA within the virion, which could further provide a roadmap for capsid assembly.
Subject(s)
Allolevivirus/ultrastructure , Capsid Proteins/chemistry , Capsid Proteins/ultrastructure , Cryoelectron Microscopy , Genome, Viral , RNA, Viral/chemistry , RNA, Viral/ultrastructure , Capsid/chemistry , Crystallography, X-Ray , EF Hand Motifs , Models, Molecular , Protein MultimerizationABSTRACT
UNLABELLED: A total of 2,691 mosquitoes representing 17 species was collected from eight locations in southwest Cameroon and screened for pathogenic viruses. Ten isolates of a novel reovirus (genus Dinovernavirus) were detected by culturing mosquito pools on Aedes albopictus (C6/36) cell cultures. A virus that caused overt cytopathic effects was isolated, but it did not infect vertebrate cells or produce detectable disease in infant mice after intracerebral inoculation. The virus, tentatively designated Fako virus (FAKV), represents the first 9-segment, double-stranded RNA (dsRNA) virus to be isolated in nature. FAKV appears to have a broad mosquito host range, and its detection in male specimens suggests mosquito-to-mosquito transmission in nature. The structure of the T=1 FAKV virion, determined to subnanometer resolution by cryoelectron microscopy (cryo-EM), showed only four proteins per icosahedral asymmetric unit: a dimer of the major capsid protein, one turret protein, and one clamp protein. While all other turreted reoviruses of known structures have at least two copies of the clamp protein per asymmetric unit, FAKV's clamp protein bound at only one conformer of the major capsid protein. The FAKV capsid architecture and genome organization represent the most simplified reovirus described to date, and phylogenetic analysis suggests that it arose from a more complex ancestor by serial loss-of-function events. IMPORTANCE: We describe the detection, genetic, phenotypic, and structural characteristics of a novel Dinovernavirus species isolated from mosquitoes collected in Cameroon. The virus, tentatively designated Fako virus (FAKV), is related to both single-shelled and partially double-shelled viruses. The only other described virus in this genus was isolated from cultured mosquito cells. It was previously unclear whether the phenotypic characteristics of that virus were reflective of this genus in nature or were altered during serial passaging in the chronically infected cell line. FAKV is a naturally occurring single-shelled reovirus with a unique virion architecture that lacks several key structural elements thought to stabilize a single-shelled reovirus virion, suggesting what may be the minimal number of proteins needed to form a viable reovirus particle. FAKV evolved from more complex ancestors by losing a genome segment and several virion proteins.
Subject(s)
Culicidae/virology , Genome, Viral , Reoviridae/genetics , Reoviridae/isolation & purification , Animals , Cameroon , Cell Line , Cluster Analysis , Cryoelectron Microscopy , Cytopathogenic Effect, Viral , Evolution, Molecular , Host Specificity , Macromolecular Substances/ultrastructure , Male , Mice , Molecular Sequence Data , Phylogeny , RNA, Viral/genetics , Reoviridae/physiology , Reoviridae/ultrastructure , Sequence Analysis, DNA , Viral Structural Proteins/ultrastructure , Virion/ultrastructure , Virus CultivationABSTRACT
Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.
Subject(s)
Bacteriophage PRD1/genetics , Bacteriophage PRD1/ultrastructure , DNA, Viral/genetics , DNA/genetics , Genome, Viral , Models, Molecular , Virus Assembly/genetics , Capsid/chemistry , Cryoelectron Microscopy , DNA, Viral/ultrastructure , Image Processing, Computer-Assisted , Membrane Proteins , Membranes , Mutation/genetics , Viral Proteins , Virion/genetics , Virion/ultrastructureABSTRACT
Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (â¼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.
Subject(s)
Bacteriophage T7/chemistry , Capsid/chemistry , Cryoelectron Microscopy , Image Processing, Computer-Assisted , Models, Molecular , Bacteriophage T7/ultrastructure , Capsid/ultrastructure , Capsid Proteins/chemistry , DNA Packaging , Protein Binding , Protein Structure, Secondary , Virus AssemblyABSTRACT
Advances in electron cryo-microscopy have enabled structure determination of macromolecules at near-atomic resolution. However, structure determination, even using de novo methods, remains susceptible to model bias and overfitting. Here we describe a complete workflow for data acquisition, image processing, all-atom modelling and validation of brome mosaic virus, an RNA virus. Data were collected with a direct electron detector in integrating mode and an exposure beyond the traditional radiation damage limit. The final density map has a resolution of 3.8 Å as assessed by two independent data sets and maps. We used the map to derive an all-atom model with a newly implemented real-space optimization protocol. The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.
Subject(s)
Bromovirus/ultrastructure , Cryoelectron Microscopy/methods , Electrons , Image Processing, Computer-Assisted/statistics & numerical data , Cryoelectron Microscopy/instrumentation , Crystallography, X-Ray , Models, Molecular , Protein ConformationABSTRACT
Orsay, the first virus discovered to naturally infect Caenorhabditis elegans or any nematode, has a bipartite, positive-sense RNA genome. Sequence analyses show that Orsay is related to nodaviruses, but molecular characterizations of Orsay reveal several unique features, such as the expression of a capsid-δ fusion protein and the use of an ATG-independent mechanism for translation initiation. Here we report the crystal structure of an Orsay virus-like particle assembled from recombinant capsid protein (CP). Orsay capsid has a T = 3 icosahedral symmetry with 60 trimeric surface spikes. Each CP can be divided into three regions: an N-terminal arm that forms an extended protein interaction network at the capsid interior, an S domain with a jelly-roll, ß-barrel fold forming the continuous capsid, and a P domain that forms surface spike projections. The structure of the Orsay S domain is best aligned to T = 3 plant RNA viruses but exhibits substantial differences compared with the insect-infecting alphanodaviruses, which also lack the P domain in their CPs. The Orsay P domain is remotely related to the P1 domain in calicivirus and hepatitis E virus, suggesting a possible evolutionary relationship. Removing the N-terminal arm produced a slightly expanded capsid with fewer nucleic acids packaged, suggesting that the arm is important for capsid stability and genome packaging. Because C. elegans-Orsay serves as a highly tractable model for studying viral pathogenesis, our results should provide a valuable structural framework for further studies of Orsay replication and infection.
Subject(s)
Caenorhabditis elegans/virology , Capsid Proteins/chemistry , Nodaviridae/chemistry , Amino Acid Sequence , Animals , Capsid Proteins/genetics , Crystallography, X-Ray , Evolution, Molecular , Molecular Sequence Data , Nodaviridae/genetics , Nodaviridae/pathogenicity , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Viral/chemistry , RNA, Viral/genetics , VirulenceABSTRACT
High-resolution structures of viruses have made important contributions to modern structural biology. Bacteriophages, the most diverse and abundant organisms on earth, replicate and infect all bacteria and archaea, making them excellent potential alternatives to antibiotics and therapies for multidrug-resistant bacteria. Here, we improved upon our previous electron cryomicroscopy structure of Salmonella bacteriophage epsilon15, achieving a resolution sufficient to determine the tertiary structures of both gp7 and gp10 protein subunits that form the T = 7 icosahedral lattice. This study utilizes recently established best practice for near-atomic to high-resolution (3-5 Å) electron cryomicroscopy data evaluation. The resolution and reliability of the density map were cross-validated by multiple reconstructions from truly independent data sets, whereas the models of the individual protein subunits were validated adopting the best practices from X-ray crystallography. Some sidechain densities are clearly resolved and show the subunit-subunit interactions within and across the capsomeres that are required to stabilize the virus. The presence of the canonical phage and jellyroll viral protein folds, gp7 and gp10, respectively, in the same virus suggests that epsilon15 may have emerged more recently relative to other bacteriophages.
Subject(s)
Models, Molecular , Salmonella Phages/chemistry , Viral Proteins/chemistry , Cryoelectron Microscopy , Crystallography, X-Ray , Protein ConformationABSTRACT
One limitation in electron cryo-microscopy (cryo-EM) is the inability to recover high-resolution signal from the image-recording media at the full-resolution limit of the transmission electron microscope. Direct electron detection using CMOS-based sensors for digitally recording images has the potential to alleviate this shortcoming. Here, we report a practical performance evaluation of a Direct Detection Device (DDD®) for biological cryo-EM at two different microscope voltages: 200 and 300 kV. Our DDD images of amorphous and graphitized carbon show strong per-pixel contrast with image resolution near the theoretical sampling limit of the data. Single-particle reconstructions of two frozen-hydrated bacteriophages, P22 and ε15, establish that the DDD is capable of recording usable signal for 3D reconstructions at about 4/5 of the Nyquist frequency, which is a vast improvement over the performance of conventional imaging media. We anticipate the unparalleled performance of this digital recording device will dramatically benefit cryo-EM for routine tomographic and single-particle structural determination of biological specimens.
Subject(s)
Cryoelectron Microscopy/methods , Bacteriophages/ultrastructure , Image Processing, Computer-AssistedABSTRACT
The eukaryotic group II chaperonin TRiC/CCT is a 16-subunit complex with eight distinct but similar subunits arranged in two stacked rings. Substrate folding inside the central chamber is triggered by ATP hydrolysis. We present five cryo-EM structures of TRiC in apo and nucleotide-induced states without imposing symmetry during the 3D reconstruction. These structures reveal the intra- and inter-ring subunit interaction pattern changes during the ATPase cycle. In the apo state, the subunit arrangement in each ring is highly asymmetric, whereas all nucleotide-containing states tend to be more symmetrical. We identify and structurally characterize an one-ring closed intermediate induced by ATP hydrolysis wherein the closed TRiC ring exhibits an observable chamber expansion. This likely represents the physiological substrate folding state. Our structural results suggest mechanisms for inter-ring-negative cooperativity, intra-ring-positive cooperativity, and protein-folding chamber closure of TRiC. Intriguingly, these mechanisms are different from other group I and II chaperonins despite their similar architecture.
Subject(s)
Adenosine Triphosphatases/metabolism , Chaperonins/chemistry , Chaperonins/metabolism , Cryoelectron Microscopy , Hydrolysis , Models, Molecular , Protein Conformation , Protein FoldingABSTRACT
Venezuelan equine encephalitis virus (VEEV), a member of the membrane-containing Alphavirus genus, is a human and equine pathogen, and has been developed as a biological weapon. Using electron cryo-microscopy (cryo-EM), we determined the structure of an attenuated vaccine strain, TC-83, of VEEV to 4.4 Å resolution. Our density map clearly resolves regions (including E1, E2 transmembrane helices and cytoplasmic tails) that were missing in the crystal structures of domains of alphavirus subunits. These new features are implicated in the fusion, assembly and budding processes of alphaviruses. Furthermore, our map reveals the unexpected E3 protein, which is cleaved and generally thought to be absent in the mature VEEV. Our structural results suggest a mechanism for the initial stage of nucleocapsid core formation, and shed light on the virulence attenuation, host recognition and neutralizing activities of VEEV and other alphavirus pathogens.