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1.  Structural Insights into the Regulatory Mechanism of the Response Regulator RocR from Pseudomonas aeruginosa in Cyclic Di-GMP Signaling 
Journal of Bacteriology  2012;194(18):4837-4846.
The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.
doi:10.1128/JB.00560-12
PMCID: PMC3430337  PMID: 22753070
2.  Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER  
Local structural similarity restraints (LSSR) provide a novel method for exploiting NCS or structural similarity to an external target structure. Two examples are given where BUSTER re-refinement of PDB entries with LSSR produces marked improvements, enabling further structural features to be modelled.
Maximum-likelihood X-ray macromolecular structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favouring NCS, or to a distinct ‘target’ structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to separate out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use -autoncs and -­target target.pdb options. The use of LSSR is illustrated in the re-refinement of PDB entries 5rnt, where -target enables the correct ligand-binding structure to be found, and 1osg, where -autoncs contributes to the location of an additional copy of the cyclic peptide ligand.
doi:10.1107/S0907444911056058
PMCID: PMC3322596  PMID: 22505257
BUSTER; NCS restraints; target-structure restraints; local structural similarity restraints
3.  Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2′-O-Methyltransferase nsp10/nsp16 Complex 
PLoS Pathogens  2011;7(5):e1002059.
Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Å2 surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.
Author Summary
A novel coronavirus emerged in 2003 and was identified as the etiological agent of the deadly disease called Severe Acute Respiratory Syndrome. This coronavirus replicates and transcribes its giant genome using sixteen non-structural proteins (nsp1-16). Viral RNAs are capped to ensure stability, efficient translation, and evading the innate immunity system of the host cell. The nsp16 protein is a RNA cap modifying enzyme only active in the presence of its activating partner nsp10. We have crystallized the nsp10/16 complex and report its crystal structure at atomic resolution. Nsp10 binds to nsp16 through a ∼930 Å2 activation surface area in nsp10, and the resulting complex exhibits RNA cap (nucleoside-2′-O)-methyltransferase activity. We have performed mutational and functional assays to identify key residues involved in catalysis and/or in RNA binding, and in the association of nsp10 to nsp16. We present two additional crystal structures, that of the known inhibitor Sinefungin bound in the SAM binding pocket, and that of a tighter complex made of the mutant nsp10(Y96F) bound to nsp16. Our study provides a basis for antiviral drug design as well as the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.
doi:10.1371/journal.ppat.1002059
PMCID: PMC3102710  PMID: 21637813
4.  Data processing and analysis with the autoPROC toolbox 
Typical topics and problems encountered during data processing of diffraction experiments are discussed and the tools provided in the autoPROC software are described.
A typical diffraction experiment will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and numbers that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concentrating on difficulties that may often show up during the first steps along the path of turning the experiment (i.e. data collection) into a model (i.e. interpreted electron density). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analysing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the experiment is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats.
doi:10.1107/S0907444911007773
PMCID: PMC3069744  PMID: 21460447
autoPROC; data processing
5.  The N-Terminal Domain of the Arenavirus L Protein Is an RNA Endonuclease Essential in mRNA Transcription 
PLoS Pathogens  2010;6(9):e1001038.
Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.
Author Summary
The Arenaviridae virus family includes several life-threatening human pathogens that cause meningitis or hemorrhagic fever. These RNA viruses replicate and transcribe their genome using an RNA synthesis machinery for which no structural data currently exist. They synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism thought to involve the large L protein, which carries RNA-dependent RNA polymerase signature sequences. Here, we report the crystal structure and functional characterization of an isolated N-terminal domain of the L protein (NL1) from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures and mutagenesis studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. Reverse genetic studies show that mutation of active site residues selectively abolish transcription, not replication. We show that this endonuclease domain is conserved and active across the virus families: Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.
doi:10.1371/journal.ppat.1001038
PMCID: PMC2940758  PMID: 20862324
6.  ‘Broken symmetries’ in macromolecular crystallography: phasing from unmerged data 
Site-specific radiation damage and anisotropy of anomalous scattering can induce intensity differences in symmetry-related reflections. If the data are kept unmerged, these symmetry-breaking effects can become a source of phase information.
The space-group symmetry of a crystal structure imposes a point-group symmetry on its diffraction pattern, giving rise to so-called symmetry-equivalent reflections. Instances in macromolecular crystallography are discussed in which the sym­metry in reciprocal space is broken, i.e. where symmetry-related reflections are no longer equivalent. Such a situation occurs when the sample suffers from site-specific radiation damage during the X-ray measurements. Another example of broken symmetry arises from the polarization anisotropy of anomalous scattering. In these cases, the genuine intensity differences between symmetry-related reflections can be exploited to yield phase information in the structure-solution process. In this approach, the usual separation of the data merging and phasing steps is abandoned. The data are kept unmerged down to the Harker construction, where the symmetry-breaking effects are explicitly modelled and refined and become a source of supplementary phase information.
doi:10.1107/S0907444909053578
PMCID: PMC2852309  PMID: 20382998
broken symmetry; phasing; radiation damage; polarization anisotropy
7.  The Crystal Structure of Coxsackievirus B3 RNA-Dependent RNA Polymerase in Complex with Its Protein Primer VPg Confirms the Existence of a Second VPg Binding Site on Picornaviridae Polymerases ▿  
Journal of Virology  2008;82(19):9577-9590.
The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3Dpol) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3Dpol at this site excludes its uridylation by the carrier 3Dpol. We suggest that VPg at this position is either uridylated by another 3Dpol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3Dpol/VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.
doi:10.1128/JVI.00631-08
PMCID: PMC2546979  PMID: 18632861
8.  Re-refinement from deposited X-ray data can deliver improved models for most PDB entries 
An evaluation of validation and real-space intervention possibilities for improving existing automated (re-)refinement methods.
The deposition of X-ray data along with the customary structural models defining PDB entries makes it possible to apply large-scale re-refinement protocols to these entries, thus giving users the benefit of improvements in X-ray methods that have occurred since the structure was deposited. Auto­mated gradient refinement is an effective method to achieve this goal, but real-space intervention is most often required in order to adequately address problems detected by structure-validation software. In order to improve the existing protocol, automated re-refinement was combined with structure validation and difference-density peak analysis to produce a catalogue of problems in PDB entries that are amenable to automatic correction. It is shown that re-refinement can be effective in producing improvements, which are often associated with the systematic use of the TLS parameterization of B factors, even for relatively new and high-resolution PDB entries, while the accompanying manual or semi-manual map analysis and fitting steps show good prospects for eventual automation. It is proposed that the potential for simultaneous improvements in methods and in re-refinement results be further encouraged by broadening the scope of depositions to include refinement metadata and ultimately primary rather than reduced X-ray data.
doi:10.1107/S0907444908037591
PMCID: PMC2631631  PMID: 19171973
re-refinement
9.  Exploiting the anisotropy of anomalous scattering boosts the phasing power of SAD and MAD experiments 
It is shown that the anisotropy of anomalous scattering (AAS) is a significant and ubiquitous effect in data sets collected at an absorption edge and that its exploitation can substantially enhance the phasing power of single- or multi-wavelength anomalous diffraction. The improvements in the phases are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment.
The X-ray polarization anisotropy of anomalous scattering in crystals of brominated nucleic acids and selenated proteins is shown to have significant effects on the diffraction data collected at an absorption edge. For conventionally collected single- or multi-wavelength anomalous diffraction data, the main manifestation of the anisotropy of anomalous scattering is the breakage of the equivalence between symmetry-related reflections, inducing intensity differences between them that can be exploited to yield extra phase information in the structure-solution process. A new formalism for describing the anisotropy of anomalous scattering which allows these effects to be incorporated into the general scheme of experimental phasing methods using an extended Harker construction is introduced. This requires a paradigm shift in the data-processing strategy, since the usual separation of the data-merging and phasing steps is abandoned. The data are kept unmerged down to the Harker construction, where the symmetry-breaking is explicitly modelled and refined and becomes a source of supplementary phase information. These ideas have been implemented in the phasing program SHARP. Refinements using actual data show that exploitation of the anisotropy of anomalous scattering can deliver substantial extra phasing power compared with conventional approaches using the same raw data. Examples are given that show improvements in the phases which are typically of the same order of magnitude as those obtained in a conventional approach by adding a second-wavelength data set to a SAD experiment. It is argued that such gains, which come essentially for free, i.e. without the collection of new data, are highly significant, since radiation damage can frequently preclude the collection of a second-wavelength data set. Finally, further developments in synchrotron instrumentation and in the design of data-collection strategies that could help to maximize these gains are outlined.
doi:10.1107/S0907444908010202
PMCID: PMC2467528  PMID: 18566507
anisotropy of anomalous scattering; phasing; SAD; MAD; polarized resonant diffraction

Results 1-9 (9)