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Biological imaging with the LCLS X-ray laser The start-up of the Linac Coherent Light Source (LCLS), the new femtosecond hard X-ray laser facility in Stanford, California, has brought high expectations of a new era for biological imaging. The intense, ultrashort X-ray pulses allow diffraction imaging of small structures before radiation damage occurs. Two papers in this issue of Nature present proof-of-concept experiments showing the LCLS in action. Chapman et al . tackle structure determination from nanocrystals of macromolecules that cannot be grown in large crystals. They obtain more than three million diffraction patterns from a stream of nanocrystals of the membrane protein photosystem I, and assemble a three-dimensional data set for this protein. Seibert et al . obtain images of a non-crystalline biological sample, mimivirus, by injecting a beam of cooled mimivirus particles into the X-ray beam. The start-up of the new femtosecond hard X-ray laser facility in Stanford, the Linac Coherent Light Source, has brought high expectations for a new era for biological imaging. The intense, ultrashort X-ray pulses allow diffraction imaging of small structures before radiation damage occurs. This new capability is tested for the problem of imaging a non-crystalline biological sample. Images of mimivirus are obtained, the largest known virus with a total diameter of about 0.75 micrometres, by injecting a beam of cooled mimivirus particles into the X-ray beam. The measurements indicate no damage during imaging and prove the concept of this imaging technique. X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions 1 , 2 , 3 , 4 . Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma 1 . The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval 2 . Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source 5 . Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.

The 2009 H1N1 swine flu is the first influenza pandemic in decades. The crystal structure of the hemagglutinin from the A/California/04/2009 H1N1 virus shows that its antigenic structure, particularly within the Sa antigenic site, is extremely similar to those of human H1N1 viruses circulating early in the 20th century. The cocrystal structure of the 1918 hemagglutinin with 2D1, an antibody from a survivor of the 1918 Spanish flu that neutralizes both 1918 and 2009 H1N1 viruses, reveals an epitope that is conserved in both pandemic viruses. Thus, antigenic similarity between the 2009 and 1918-like viruses provides an explanation for the age-related immunity to the current influenza pandemic.

Influenza virus: polymerase structures determined Influenza A virus polymerase consists of three proteins, PA, PB1 and PB2, and is critical for transcription and replication. Two groups now report the crystal structure of the avian H5N1 PA C-terminal domain in complex with the PA binding domain of PB1. The structure work may be useful for the design of novel antiviral drugs. The recent emergence of highly pathogenic avian influenza A virus strains with subtype H5N1 pose a global threat to human health 1 . Elucidation of the underlying mechanisms of viral replication is critical for development of anti-influenza virus drugs 2 . The influenza RNA-dependent RNA polymerase (RdRp) heterotrimer has crucial roles in viral RNA replication and transcription. It contains three proteins: PA, PB1 and PB2. PB1 harbours polymerase and endonuclease activities and PB2 is responsible for cap binding 3 , 4 ; PA is implicated in RNA replication 5 , 6 , 7 , 8 , 9 , 10 and proteolytic activity 11 , 12 , 13 , 14 , although its function is less clearly defined. Here we report the 2.9 ångström structure of avian H5N1 influenza A virus PA (PA C , residues 257–716) in complex with the PA-binding region of PB1 (PB1 N , residues 1–25). PA C has a fold resembling a dragon’s head with PB1 N clamped into its open ‘jaws’. PB1 N is a known inhibitor that blocks assembly of the polymerase heterotrimer and abolishes viral replication. Our structure provides details for the binding of PB1 N to PA C at the atomic level, demonstrating a potential target for novel anti-influenza therapeutics. We also discuss a potential nucleotide binding site and the roles of some known residues involved in polymerase activity. Furthermore, to explore the role of PA in viral replication and transcription, we propose a model for the influenza RdRp heterotrimer by comparing PA C with the λ3 reovirus polymerase structure, and docking the PA C structure into an available low resolution electron microscopy map.

Construction of a complex virus may involve a hierarchy of assembly elements. Here, we report the structure of the whole human adenovirus virion at 3.6 angstroms resolution by cryo-electron microscopy (cryo-EM), revealing in situ atomic models of three minor capsid proteins (IIIa, VIII, and IX), extensions of the (penton base and hexon) major capsid proteins, and interactions within three protein-protein networks. One network is mediated by protein IIIa at the vertices, within group-of-six (GOS) tiles--a penton base and its five surrounding hexons. Another is mediated by ropes (protein IX) that lash hexons together to form group-of-nine (GON) tiles and bind GONs to GONs. The third, mediated by IIIa and VIII, binds each GOS to five surrounding GONs. Optimization of adenovirus for cancer and gene therapy could target these networks.

Influenza changes channels Until recently, the pH-gated proton channel of influenza A virus, M2, was effectively targeted by amantadine-based antivirals, but resistance to these drugs is now widespread. Two groups now present structural studies of M2 proton channel. Jason Schnell and James Chou determine the structure of a 38-residue segment of M2, in complex with rimantadine, by NMR spectroscopy. Amanda Stouffer et al . determined the crystal structure of a 25-residue fragment of M2, with and without amantadine, using X-ray diffraction. Strikingly, the resulting structures suggest two very different mechanisms by which the drug inhibits the channel. The proposed mechanisms are discussed by Christopher Miller in an accompanying News & Views article. A vital component of influenza A virus' replication machinery is the M2 proton channel. Until recently, M2 was effectively targeted by amantadane-based antivirals, but resistance to these drugs is now so widespread that they have become ineffective. In the first of two related papers, the structure of a 38-residue segment of M2, in complex with rimantadine, is determined by NMR spectroscopy. It is concluded that a rimantadine molecule binds to each monomer at the protein–lipid interface and inhibits the tetrameric channel allosterically. The integral membrane protein M2 of influenza virus forms pH-gated proton channels in the viral lipid envelope 1 . The low pH of an endosome activates the M2 channel before haemagglutinin-mediated fusion. Conductance of protons acidifies the viral interior and thereby facilitates dissociation of the matrix protein from the viral nucleoproteins—a required process for unpacking of the viral genome 2 . In addition to its role in release of viral nucleoproteins, M2 in the trans-Golgi network (TGN) membrane prevents premature conformational rearrangement of newly synthesized haemagglutinin during transport to the cell surface by equilibrating the pH of the TGN with that of the host cell cytoplasm 3 . Inhibiting the proton conductance of M2 using the anti-viral drug amantadine or rimantadine inhibits viral replication 4 , 5 , 6 , 7 . Here we present the structure of the tetrameric M2 channel in complex with rimantadine, determined by NMR. In the closed state, four tightly packed transmembrane helices define a narrow channel, in which a ‘tryptophan gate’ is locked by intermolecular interactions with aspartic acid. A carboxy-terminal, amphipathic helix oriented nearly perpendicular to the transmembrane helix forms an inward-facing base. Lowering the pH destabilizes the transmembrane helical packing and unlocks the gate, admitting water to conduct protons, whereas the C-terminal base remains intact, preventing dissociation of the tetramer. Rimantadine binds at four equivalent sites near the gate on the lipid-facing side of the channel and stabilizes the closed conformation of the pore. Drug-resistance mutations are predicted to counter the effect of drug binding by either increasing the hydrophilicity of the pore or weakening helix–helix packing, thus facilitating channel opening.

HIV/AIDS: gp120 structure changes This paper investigates the structure of the HIV the gp120 coat protein of HIV by cryo-electron tomography and molecular modelling. Comparison of gp120 structures in an unbound state, bound to a neutralizing antibody and bound to CD4 cell surface protein provides insight into the conformational changes that occur during antibody neutralization and attachment to target cells. This paper investigates the structure of the HIV glycoprotein gp120 by cryo-electron tomography and molecular modelling. gp120 is analysed in an unliganded state, complexed with a neutralizing antibody and in a CD4 liganded state. The analysis provides insight into the conformational changes that occur with ligand binding. The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection 1 . Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with three-dimensional image classification and averaging, we report the three-dimensional structures of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures 2 , 3 of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells.

Avian flu virus polymerase The influenza virus RNA-dependent RNA polymerase, which contains three subunits (PA, PB1 and PB2), directs the replication and transcription of viral RNA inside the nuclei of infected cells. Two groups now report the crystal structure of the N terminus of the PA subunit of avian influenza virus. Structural comparison and mutational analysis show that the PA subunit contains an endonucleolytic cleavage site, an activity previously suspected to reside on the PB1 subunit. The PA endonuclease active site is highly conserved across influenza strains, making it a promising potential target for new anti-influenza drugs. This paper reports the crystal structure of the amino terminus of the PA subunit of the influenza RNA polymerase, and provides evidence that it has endonuclease activity. The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site 1 and reportedly harbours endonuclease activity 2 , whereas PB2 is responsible for cap binding 2 , 3 , 4 . The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease 5 and protease activities 6 as well as viral RNA/complementary RNA promoter binding 7 . Here we report the 2.2 ångström (Å) crystal structure of the N-terminal 197 residues of PA, termed PA N , from an avian influenza H5N1 virus. The PA N structure has an α/β architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX N (D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA N provide further evidence that PA N holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA N holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA N is an important target for the design of new anti-influenza therapeutics.

Avian flu virus polymerase The influenza virus RNA-dependent RNA polymerase, which contains three subunits (PA, PB1 and PB2), directs the replication and transcription of viral RNA inside the nuclei of infected cells. Two groups now report the crystal structure of the N terminus of the PA subunit of avian influenza virus. Structural comparison and mutational analysis show that the PA subunit contains an endonucleolytic cleavage site, an activity previously suspected to reside on the PB1 subunit. The PA endonuclease active site is highly conserved across influenza strains, making it a promising potential target for new anti-influenza drugs. The amino terminal domain of influenza virus polymerase PA subunit is shown to harbour the endonuclease activity required for the cap-snatching mechanism of viral mRNA synthesis. The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism 1 . The PB2 subunit binds the 5′ cap of host pre-mRNAs 2 , 3 , 4 , which are subsequently cleaved after 10–13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5 ) or PB1 (ref. 2 ) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase 6 , 7 . Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease 8 . The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated 9 . These results will facilitate the optimisation of endonuclease inhibitors 10 , 11 , 12 as potential new anti-influenza drugs.

The respiratory syncytial virus (RSV) is an important human pathogen, yet neither a vaccine nor effective therapies are available to treat infection. To help elucidate the replication mechanism of this RNA virus, we determined the three-dimensional (3D) crystal structure at 3.3 Å resolution of a decameric, annular ribonucleoprotein complex of the RSV nucleoprotein (N) bound to RNA. This complex mimics one turn of the viral helical nucleocapsid complex, which serves as template for viral RNA synthesis. The RNA wraps around the protein ring, with seven nucleotides contacting each N subunit, alternating rows of four and three stacked bases that are exposed and buried within a protein groove, respectively. Combined with electron microscopy data, this structure provides a detailed model for the RSV nucleocapsid, in which the bases are accessible for readout by the viral polymerase. Furthermore, the nucleoprotein structure highlights possible key sites for drug targeting.

Intracellular cleavage of immature flaviviruses is a critical step in assembly that generates the membrane fusion potential of the E glycoprotein. With cryo--electron microscopy we show that the immature dengue particles undergo a reversible conformational change at low pH that renders them accessible to furin cleavage. At a pH of 6.0, the E proteins are arranged in a herringbone pattern with the pr peptides docked onto the fusion loops, a configuration similar to that of the mature virion. After cleavage, the dissociation of pr is pH-dependent, suggesting that in the acidic environment of the trans-Golgi network pr is retained on the virion to prevent membrane fusion. These results suggest a mechanism by which flaviviruses are processed and stabilized in the host cell secretory pathway.