Structure and function of Plasmodium actin II in the parasite mosquito stages.

Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and also have nuclear functions. Malaria parasites (Plasmodium spp.

Electron cryotomography of SARS-CoV-2 virions reveals cylinder-shaped particles with a double layer RNP assembly.

SARS-CoV-2 is a lipid-enveloped Betacoronavirus and cause of the Covid-19 pandemic. To study the three-dimensional architecture of the virus, we perform electron cryotomography (cryo-ET) on SARS-Cov-2 virions and three variants revealing particles of regular cylindrical morphology. The ribonucleoprotein particles packaging the genome in the virion interior form a dense, double layer assembly with a cylindrical shape related to the overall particle morphology.

Cryomicroscopy reveals the structural basis for a flexible hinge motion in the immunoglobulin M pentamer.

Immunoglobulin M (IgM) is the most ancient of the five isotypes of immunoglobulin (Ig) molecules and serves as the first line of defence against pathogens. Here, we use cryo-EM to image the structure of the human full-length IgM pentamer, revealing antigen binding domains flexibly attached to the asymmetric and rigid core formed by the Cμ4 and Cμ3 constant regions and the J-chain. A hinge is located at the Cμ3/Cμ2 domain interface, allowing Fabs and Cμ2 to pivot as a unit both in-plane and out-of-plane.

Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH.

The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface-bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA.

High-resolution structures of malaria parasite actomyosin and actin filaments.

Malaria is responsible for half a million deaths annually and poses a huge economic burden on the developing world. The mosquito-borne parasites (Plasmodium spp.) that cause the disease depend upon an unconventional actomyosin motor for both gliding motility and host cell invasion.

In situ structure and organization of the influenza C virus surface glycoprotein.

The lipid-enveloped influenza C virus contains a single surface glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, that mediates receptor binding, receptor destruction, and membrane fusion at the low pH of the endosome. Here we apply electron cryotomography and subtomogram averaging to describe the structural basis for hexagonal lattice formation by HEF on the viral surface. The conformation of the glycoprotein in situ is distinct from the structure of the isolated trimeric ectodomain, showing that a splaying of the membrane distal domains is required to mediate contacts that form the lattice.

Structure and binding properties of Pangolin-CoV spike glycoprotein inform the evolution of SARS-CoV-2.

Coronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.

Influenza hemagglutinin membrane anchor.

Viruses with membranes fuse them with cellular membranes, to transfer their genomes into cells at the beginning of infection. For Influenza virus, the membrane glycoprotein involved in fusion is the hemagglutinin (HA), the 3D structure of which is known from X-ray crystallographic studies. The soluble ectodomain fragments used in these studies lacked the "membrane anchor" portion of the molecule.

Three-Dimensional Architecture of the Human BRCA1-A Histone Deubiquitinase Core Complex.

BRCA1 is a tumor suppressor found to be mutated in hereditary breast and ovarian cancer and plays key roles in the maintenance of genomic stability by homologous recombination repair. It is recruited to damaged chromatin as a component of the BRCA1-A deubiquitinase, which cleaves K63-linked ubiquitin chains attached to histone H2A and H2AX. BRCA1-A contributes to checkpoint regulation, repair pathway choice, and HR repair efficiency through molecular mechanisms that remain largely obscure.

Cryomicroscopy provides structural snapshots of influenza virus membrane fusion.

The lipid-enveloped influenza virus enters host cells during infection by binding cell-surface receptors and, after receptor-mediated endocytosis, fusing with the membrane of the endosome and delivering the viral genome and transcription machinery into the host cell. These events are mediated by the hemagglutinin (HA) surface glycoprotein. At the low pH of the endosome, an irreversible conformational change in the HA, including the exposure of the hydrophobic fusion peptide, activates membrane fusion.

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody.

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo.

Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation.

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV_F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif.

Structural studies of postentry restriction factors reveal antiparallel dimers that enable avid binding to the HIV-1 capsid lattice.

Restriction factors (RFs) form important components of host defenses to retroviral infection. The Fv1, Trim5α, and TrimCyp RFs contain N-terminal dimerization and C-terminal specificity domains that target assembled retroviral capsid (CA) proteins enclosing the viral core. However, the molecular detail of the interaction between RFs and their CA targets is unknown.

Cryomicroscopy of radiation sensitive specimens on unmodified graphene sheets: reduction of electron-optical effects of charging.

Images of radiation-sensitive specimens obtained by electron microscopy suffer a reduction in quality beyond that expected from radiation damage alone due to electron beam-induced charging or movement of the specimen. For biological specimens, charging and movement are most severe when they are suspended in an insulating layer of vitreous ice, which is otherwise optimal for preserving hydrated specimens in a near native state. We image biological specimens, including a single particle protein complex and a lipid-enveloped virus in thin, vitreous ice films over suspended sheets of unmodified graphene.

Distribution of surface glycoproteins on influenza A virus determined by electron cryotomography.

We use electron cryotomography to reconstruct virions of two influenza A H3N2 virus strains. The maps reveal the structure of the viral envelope containing hemagglutinin (HA) and neuraminidase (NA) glycoproteins and the virus interior containing a matrix layer and an assembly of ribonucleoprotein particles (RNPs) that package the genome. We build a structural model for the viral surface by locating copies of the X-ray structure of the HA ectodomain into density peaks on the virus surface.

A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses.

Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses.

A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins.

The isolation of broadly neutralizing antibodies against influenza A viruses has been a long-sought goal for therapeutic approaches and vaccine design. Using a single-cell culture method for screening large numbers of human plasma cells, we isolated a neutralizing monoclonal antibody that recognized the hemagglutinin (HA) glycoprotein of all 16 subtypes and neutralized both group 1 and group 2 influenza A viruses. Passive transfer of this antibody conferred protection to mice and ferrets.

Malaria parasite actin polymerization and filament structure.

A novel form of acto-myosin regulation has been proposed in which polymerization of new actin filaments regulates motility of parasites of the apicomplexan class of protozoa. In vivo and in vitro parasite F-actin is very short and unstable, but the structural basis and details of filament dynamics remain unknown. Here, we show that long actin filaments can be obtained by polymerizing unlabeled rabbit skeletal actin (RS-actin) onto both ends of the short rhodamine-phalloidin-stabilized Plasmodium falciparum actin I (Pf-actin) filaments.

Functional interactions as a survival strategy against abnormal aggregation.

Protein aggregation is under intense scrutiny because of its role in human disease. Although increasing evidence indicates that protein native states are highly protected against aggregation, the specific protection mechanisms are poorly understood. Insight into such mechanisms can be gained through study of the relatively few proteins that aggregate under native conditions.

Structural organization of a filamentous influenza A virus.

Influenza is a lipid-enveloped, pleomorphic virus. We combine electron cryotomography and analysis of images of frozen-hydrated virions to determine the structural organization of filamentous influenza A virus. Influenza A/Udorn/72 virions are capsule-shaped or filamentous particles of highly uniform diameter.

Structural analysis reveals an amyloid form of the human papillomavirus type 16 E1--E4 protein and provides a molecular basis for its accumulation.

The abundant human papillomavirus (HPV) type 16 E4 protein exists as two distinct structural forms in differentiating epithelial cells. Monomeric full-length 16E1--E4 contains a limited tertiary fold constrained by the N and C termini. N-terminal deletions facilitate the assembly of E1--E4 into amyloid-like fibrils, which bind to thioflavin T.

Structural properties of the human respiratory syncytial virus P protein: evidence for an elongated homotetrameric molecule that is the smallest orthologue within the family of paramyxovirus polymerase cofactors.

The oligomeric state and the hydrodynamic properties of human respiratory syncytial virus (HRSV) phosphoprotein (P), a known cofactor of the viral RNA-dependent RNA polymerase (L), and a trypsin-resistant fragment (X) that includes its oligomerization domain were analyzed by sedimentation equilibrium and velocity using analytical ultracentrifugation. The results obtained demonstrate that both P and fragment X are homotetrameric with elongated shapes, consistent with electron micrographs of the purified P protein in which thin rod-like molecules of approximately 12.5 +/- 1.

High level expression of soluble glycoproteins in the allantoic fluid of embryonated chicken eggs using a Sendai virus minigenome system.

Background: Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now describe a system, based on a Sendai virus minigenome, to produce large amounts of heterologous viral glycoproteins in the allantoic cavity of embryonated eggs.

Sequence elements of the fusion peptide of human respiratory syncytial virus fusion protein required for activity.

We have reported previously the expression and purification of an anchorless form of the human respiratory syncytial virus (HRSV) F protein (F(TM-)) representing the ectodomain of the full-length F. F(TM-) molecules are seen as unaggregated cones by electron microscopy but completion of proteolytic cleavage of the F0 monomers in the F(TM-) trimer leads to a change in shape from cones to lollipops that aggregate into rosettes. This aggregation apparently occurs by interaction of the fusion peptides of F(TM-) molecules that are exposed after cleavage.