Publications (18)181.85 Total impact
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Article: Visualizing a Complete Siphoviridae by Single-particle Electron Microscopy: The Structure of Lactococcal Phage TP901-1.
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ABSTRACT: Tailed phages are genome delivery machines exhibiting an unequaled efficiency acquired over more than 3 billion years of evolution. Siphophages from the P335 and 936 families infect the Gram(+) bacterium Lactococcus lactis using receptor-binding proteins anchored to the host-adsorption apparatus (baseplate). Crystallographic and electron microscopy studies have shed light on the distinct adsorption strategies used by phages of these two families suggesting that they might also rely on different infection mechanisms. We report here electron microscopy reconstructions of the whole phage TP901-1 (P335-species) and propose a composite EM model of this gigantic molecular machine. Our results suggest conservation of structural proteins among tailed phages and add to the growing body of evidence pointing to a common evolutionary origin for these virions. Finally, we propose that host-adsorption apparatus architectures have evolved in correlation with the nature of the receptors used during infection.Journal of Virology 11/2012; · 5.40 Impact Factor -
Article: Structure and molecular assignment of lactococcal phage TP901-1 baseplate.
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ABSTRACT: P335 lactococcal phages infect the gram(+) bacterium Lactococcus lactis using a large multiprotein complex located at the distal part of the tail and termed baseplate (BP). The BP harbors the receptor-binding proteins (RBPs), which allow the specific recognition of saccharidic receptors localized on the host cell surface. We report here the electron microscopic structure of the phage TP901-1 wild-type BP as well as those of two mutants bppL (-) and bppU(-), lacking BppL (the RBPs) or both peripheral BP components (BppL and BppU), respectively. We also achieved an electron microscopic reconstruction of a partial BP complex, formed by BppU and BppL. This complex exhibits a tripod shape and is composed of nine BppLs and three BppUs. These structures, combined with light-scattering measurements, led us to propose that the TP901-1 BP harbors six tripods at its periphery, located around the central tube formed by ORF46 (Dit) hexamers, at its proximal end, and a ORF47 (Tal) trimer at its distal extremity. A total of 54 BppLs (18 RBPs) are thus available to mediate host anchoring with a large apparent avidity. TP901-1 BP exhibits an infection-ready conformation and differs strikingly from the lactococcal phage p2 BP, bearing only 6 RBPs, and which needs a conformational change to reach its activated state. The comparison of several Siphoviridae structures uncovers a close organization of their central BP core whereas striking differences occur at the periphery, leading to diverse mechanisms of host recognition.Journal of Biological Chemistry 10/2010; 285(50):39079-86. · 4.77 Impact Factor -
Article: Structure of lactococcal phage p2 baseplate and its mechanism of activation
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ABSTRACT: Siphoviridae is the most abundant viral family on earth which infects bacteria as well as archaea. All known siphophages infecting gram+ Lactococcus lactis possess a baseplate at the tip of their tail involved in host recognition and attachment. Here, we report analysis of the p2 phage baseplate structure by X-ray crystallography and electron microscopy and propose a mechanism for the baseplate activation during attachment to the host cell. This ∼1 MDa, Escherichia coli-expressed baseplate is composed of three protein species, including six trimers of the receptor-binding protein (RBP). RBPs host-recognition domains point upwards, towards the capsid, in agreement with the electron-microscopy map of the free virion. In the presence of Ca2+, a cation mandatory for infection, the RBPs rotated 200° downwards, presenting their binding sites to the host, and a channel opens at the bottom of the baseplate for DNA passage. These conformational changes reveal a novel siphophage activation and host-recognition mechanism leading ultimately to DNA ejection.Proceedings of the National Academy of Sciences 04/2010; 107(15):6852-6857. · 9.68 Impact Factor -
Article: Molecular architecture of the "stressosome," a signal integration and transduction hub.
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ABSTRACT: A commonly used strategy by microorganisms to survive multiple stresses involves a signal transduction cascade that increases the expression of stress-responsive genes. Stress signals can be integrated by a multiprotein signaling hub that responds to various signals to effect a single outcome. We obtained a medium-resolution cryo-electron microscopy reconstruction of the 1.8-megadalton "stressosome" from Bacillus subtilis. Fitting known crystal structures of components into this reconstruction gave a pseudoatomic structure, which had a virus capsid-like core with sensory extensions. We suggest that the different sensory extensions respond to different signals, whereas the conserved domains in the core integrate the varied signals. The architecture of the stressosome provides the potential for cooperativity, suggesting that the response could be tuned dependent on the magnitude of chemophysical insult.Science 11/2008; 322(5898):92-6. · 31.20 Impact Factor -
Article: Structural studies of the archaeal MCM complex in different functional states.
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ABSTRACT: The primary candidate for the eukaryotic replicative helicase is the MCM2-7 complex, a hetero-oligomer formed by six AAA+ paralogous polypeptides. A simplified model for structure-function studies is the homo-oligomeric orthologue from the archaeon Methanothermobacter thermoautotrophicus. The crystal structure of the DNA-interacting N-terminal domain of this homo-oligomer revealed a double hexamer in a head-to-head configuration; single-particle electron microscopy studies have shown that the full-length protein complex can form both single and double rings, in which each ring can consist of a cyclical arrangement of six or seven subunits. Using single-particle techniques and especially multivariate statistical symmetry analysis, we have assessed the changes in stoichiometry that the complex undergoes when treated with various nucleotide analogues or when binding a double-stranded DNA fragment. We found that the binding of nucleotides or of double-stranded DNA leads to the preferred formation of double-ring structures. Specifically, the protein complex is present as a double heptamer when treated with a nucleotide analogue, but it is rather found as a double hexamer when complexed with double-stranded DNA. The possible physiological role of the various stoichiometries of the complex is discussed in the light of the proposed mechanisms of helicase activity.Journal of Structural Biology 11/2006; 156(1):210-9. · 3.41 Impact Factor -
Article: Structural basis of the Methanothermobacter thermautotrophicus MCM helicase activity.
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ABSTRACT: The MCM complex from the archaeon Methanother-mobacter thermautotrophicus is a model for the eukaryotic MCM2-7 helicase. We present electron-microscopy single-particle reconstructions of a DNA treated M.thermautotrophicus MCM sample and a ADP.AlF(x) treated sample, respectively assembling as double hexamers and double heptamers. The electron-density maps display an unexpected asymmetry between the two rings, suggesting that large conformational changes can occur within the complex. The structure of the MCM N-terminal domain, as well as the AAA+ and the C-terminal HTH dom-ains of ZraR can be fitted into the reconstructions. Distinct configurations can be modelled for the AAA+ and the HTH domains, suggesting the nature of the conformational change within the complex. The pre-sensor 1 and the helix 2 insertions, important for the activity, can be located pointing towards the centre of the channel in the presence of DNA. We propose a mechanistic model for the helicase activity, based on a ligand-controlled rotation of the AAA+ subunits.Nucleic Acids Research 02/2006; 34(20):5829-38. · 8.03 Impact Factor -
Article: Conformational changes of Escherichia coli sigma54-RNA-polymerase upon closed-promoter complex formation.
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ABSTRACT: RNA polymerase from the mesophile Escherichia coli exists in two forms, the core enzyme and the holoenzyme. Using cryo-electron microscopy and single-particle analysis, we have obtained the structure of the complete RNA polymerase from E.coli containing the sigma54 factor within the closed-promoter complex. Comparisons with earlier reconstructions of the core enzyme and the sigma54 holoenzyme reveal the behaviour of this major variant RNA polymerase in defined functional states. The binding of DNA leads to significant conformational changes in the enzyme's catalytic subunits, apparently a necessity for the initiation of enhancer-dependent promoter-specific transcription.Journal of Molecular Biology 12/2005; 354(2):201-5. · 4.00 Impact Factor -
Article: Magnification variations due to illumination curvature and object defocus in transmission electron microscopy.
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ABSTRACT: It has previously been shown that - in theory - magnification variations can occur in an imaging system as a function of defocus, depending on the field curvature of the illuminating system. We here present the results of practical experiments to verify this effect in the transmission electron microscope. We find that with illumination settings typically used in the electron microscopy of biological macromolecules, systematic variations in magnification of ~ 0.5% per microm defocus can easily occur. This work highlights the need for a magnification-invariant imaging mode to eliminate or to compensate for this effect.Optics Express 11/2005; 13(22):9085-93. · 3.59 Impact Factor -
Article: Fourier shell correlation threshold criteria.
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ABSTRACT: The resolution value claimed for an electron microscopical three-dimensional reconstruction indicates the overall quality of the experiment. The Fourier shell correlation (FSC) criterion has now become the standard quality measure. However, what has continued to be controversial is the issue of the FSC threshold level at which one defines the reproducible resolution. Here, we discuss the theoretical behaviour of the FSC in conjunction with the various factors which influence it: the number of "voxels" in a given Fourier shell, the symmetry of the structure, and the size of the structure within the reconstruction volume. Both the theoretical considerations and our model experiments show that fixed-valued FSC threshold (like "0.5") may never be used in a reproducible criterion. Fixed threshold values are-as we show here-simply the result of incorrect assumptions in the basic statistics. Two families of FSC threshold curves are discussed: the sigma-factor curves and the new family of bit-based information threshold curves. Whereas sigma-factor curves indicate the resolution level at which one has collected information significantly above the noise level, the information curves indicate the resolution level at which enough information has been collected for interpretation.Journal of Structural Biology 10/2005; 151(3):250-62. · 3.41 Impact Factor -
Article: Visualization of release factor 3 on the ribosome during termination of protein synthesis.
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ABSTRACT: Termination of protein synthesis by the ribosome requires two release factor (RF) classes. The class II RF3 is a GTPase that removes class I RFs (RF1 or RF2) from the ribosome after release of the nascent polypeptide. RF3 in the GDP state binds to the ribosomal class I RF complex, followed by an exchange of GDP for GTP and release of the class I RF. As GTP hydrolysis triggers release of RF3 (ref. 4), we trapped RF3 on Escherichia coli ribosomes using a nonhydrolysable GTP analogue. Here we show by cryo-electron microscopy that the complex can adopt two different conformational states. In 'state 1', RF3 is pre-bound to the ribosome, whereas in 'state 2' RF3 contacts the ribosome GTPase centre. The transfer RNA molecule translocates from the peptidyl site in state 1 to the exit site in state 2. This translocation is associated with a large conformational rearrangement of the ribosome. Because state 1 seems able to accommodate simultaneously both RF3 and RF2, whose position is known from previous studies, we can infer the release mechanism of class I RFs.Nature 03/2004; 427(6977):862-5. · 36.28 Impact Factor -
Article: Hexameric ring structure of the full-length archaeal MCM protein complex.
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ABSTRACT: In eukaryotes, a family of six homologous minichromosome maintenance (MCM) proteins has a key function in ensuring that DNA replication occurs only once before cell division. Whereas all eukaryotes have six paralogues, in some Archaea a single protein forms a homomeric assembly. The complex is likely to function as a helicase during DNA replication. We have used electron microscopy to obtain a three-dimensional reconstruction of the full-length MCM from Methanobacterium thermoautotrophicum. Six monomers are arranged around a sixfold axis, generating a ring-shaped molecule with a large central cavity and lateral holes. The channel running through the molecule can easily accommodate double-stranded DNA. The crystal structure of the amino-terminal fragment of MCM and a model for the AAA+ hexamer have been docked into the map, whereas additional electron density suggests that the carboxy-terminal domain is located at the interface between the two domains. The structure suggests that the MCM complex is likely to act in a different manner to traditional hexameric helicases and is likely to bear more similarity to the SV40 large T antigen or to double-stranded DNA translocases.EMBO Reports 12/2003; 4(11):1079-83. · 7.36 Impact Factor -
Article: Structure of a viral DNA gatekeeper at 10 A resolution by cryo-electron microscopy.
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ABSTRACT: In tailed bacteriophages and herpes viruses, the viral DNA is packaged through the portal protein channel. Channel closure is essential to prevent DNA release after packaging. Here we present the connector structure from bacteriophage SPP1 using cryo-electron microscopy and single particle analysis. The multiprotein complex comprises the portal protein gp6 and the head completion proteins gp15 and gp16. Although we show that gp6 in the connector has a fold similar to that of the isolated portal protein, we observe conformational changes in the region of gp6 exposed to the DNA-packaging ATPase and to gp15. This reorganization does not cause closure of the channel. The connector channel traverses the full height of gp6 and gp15, but it is closed by gp16 at the bottom of the complex. Gp16 acts as a valve whose closure prevents DNA leakage, while its opening is required for DNA release upon interaction of the virus with its host.The EMBO Journal 04/2003; 22(6):1255-62. · 9.20 Impact Factor -
Article: Voltage-gated K+ channel from mammalian brain: 3D structure at 18A of the complete (alpha)4(beta)4 complex.
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ABSTRACT: Voltage-sensitive K(+) channels (Kv) serve numerous important roles, e.g. in the control of neuron excitability and the patterns of synaptic activity. Here, we use electron microscopy (EM) and single particle analysis to obtain the first, complete structure of Kv1 channels, purified from rat brain, which contain four transmembrane channel-forming alpha-subunits and four cytoplasmically-associated beta-subunits. The 18A resolution structure reveals an asymmetric, dumb-bell-shaped complex with 4-fold symmetry, a length of 140A and variable width. By fitting published X-ray data for recombinant components to our EM map, the modulatory (beta)(4) was assigned to the innermost 105A end, the N-terminal (T1)(4) domain of the alpha-subunit to the central 50A moiety and the pore-containing portion to the 125A membrane part. At this resolution, the selectivity filter could not be localised. Direct contact of the membrane component with the central (T1)(4) domain occurs only via peripheral connectors, permitting communication between the channel and beta-subunits for coupling of responses to changes in excitability and metabolic status of neurons.Journal of Molecular Biology 03/2003; 326(4):1005-12. · 4.00 Impact Factor -
Article: Determination of Escherichia coli RNA polymerase structure by single particle cryoelectron microscopy.
Methods in Enzymology 02/2003; 370:24-42. · 2.04 Impact Factor -
Article: Structure of the Escherichia coli ribosomal termination complex with release factor 2.
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ABSTRACT: Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.Nature 02/2003; 421(6918):90-4. · 36.28 Impact Factor -
Article: Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-recognition complex.
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ABSTRACT: The mRNA codon in the ribosomal A-site is recognized by aminoacyl-tRNA (aa-tRNA) in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here we report the 13 A resolution three-dimensional reconstruction determined by cryo-electron microscopy of the kirromycin-stalled codon-recognition complex. The structure of the ternary complex is distorted by binding of the tRNA anticodon arm in the decoding center. The aa-tRNA interacts with 16S rRNA, helix 69 of 23S rRNA and proteins S12 and L11, while the sarcin-ricin loop of 23S rRNA contacts domain 1 of EF-Tu near the nucleotide-binding pocket. These results provide a detailed snapshot view of an important functional state of the ribosome and suggest mechanisms of decoding and GTPase activation.Natural Structural Biology 12/2002; 9(11):849-54. -
Article: Structural organisation of the head-to-tail interface of a bacterial virus
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ABSTRACT: In tailed icosahedral bacteriophages the connection between the 5-fold symmetric environment of the portal vertex in the capsid and the 6-fold symmetric phage tail is formed by a complex interface structure. The current study provides the detailed analysis of the assembly and structural organisation of such an interface within a phage having a long tail. The region of the interface assembled as part of the viral capsid (connector) was purified from DNA-filled capsids of the Bacillus subtilis bacteriophage SPP1. It is composed of oligomers of gp6, the SPP1 portal protein, of gp15, and of gp16. The SPP1 connector structure is formed by a mushroom-like portal protein whose cap faces the interior of the viral capsid in intact virions, an annular structure below the stem of the mushroom, and a second narrower annulus that is in direct contact with the helical tail extremity. The layered arrangement correlates to the stacking of gp6, gp15, and gp16 on top of the tail. The gp16 ring is exposed to the virion outside. During SPP1 morphogenesis, gp6 participates in the procapsid assembly reaction, an early step in the assembly pathway, while gp15 and gp16 bind to the capsid portal vertex after viral chromosome encapsidation. gp16 is processed during or after tail attachment to the connector region. The portal protein gp6 has 12-fold cyclical symmetry in the connector structure, whereas assembly-naı̈ve gp6 exhibits 13-fold symmetry. We propose that it is the interaction of gp6 with other viral morphogenetic proteins that drives its assembly into the 12-mer state.Journal of Molecular Biology 08/2001; · 4.00 Impact Factor -
Article: Escherichia coli RNA polymerase core and holoenzyme structures
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ABSTRACT: Multisubunit RNA polymerase is an essential enzyme for regulated gene expression. Here we report two Escherichia coli RNA polymerase structures: an 11.0 Å structure of the core RNA polymerase and a 9.5 Å structure of the 70 holoenzyme. Both structures were obtained by cryo-electron microscopy and angular reconstitution. Core RNA polymerase exists in an open conformation. Extensive conformational changes occur between the core and the holoenzyme forms of the RNA polymerase, which are largely associated with movements in '. All common RNA polymerase subunits (2, , ') could be localized in both structures, thus suggesting the position of 70 in the holoenzyme.The EMBO Journal 12/2000; 19(24):6833-6844. · 9.20 Impact Factor
Top co-authors
Top Journals
Institutions
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2000–2012
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Imperial College London
- • Division of Molecular Biosciences
- • Division of Cell and Molecular Biology
London, ENG, United Kingdom
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2010
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Architecture et Fonction des Macromolécules Biologiques
Marseille, Provence-Alpes-Cote d'Azur, France
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2002
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Max-Planck-Institut für biophysikalische Chemie
Göttingen, Lower Saxony, Germany
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