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1.  Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) from Neisseria meningitidis  
The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator, was solved at 2.5 Å resolution.
The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from Neisseria meningitidis, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators.
doi:10.1107/S1744309112010603
PMCID: PMC3388910  PMID: 22750853
MetR; Neisseria meningitidis; LysR-type regulator
2.  In cellulo structure determination of a novel cypovirus polyhedrin 
The crystal structure of a previously unsolved type of cypovirus polyhedrin has been determined from data collected directly from frozen live insect cells.
This work demonstrates that with the use of a microfocus synchrotron beam the structure of a novel viral polyhedrin could be successfully determined from microcrystals within cells, removing the preparatory step of sample isolation and maintaining a favourable biological environment. The data obtained are of high quality, comparable to that obtained from isolated crystals, and enabled a facile structure determination. A small but significant difference is observed between the unit-cell parameters and the mosaic spread of in cellulo and isolated crystals, suggesting that even these robust crystals are adversely affected by removal from the cell.
doi:10.1107/S1399004714004714
PMCID: PMC4014125  PMID: 24816111
microcrystals; viral protein; data collection; in cellulo
3.  Exploiting fast detectors to enter a new dimension in room-temperature crystallography 
A departure from a linear or an exponential decay in the diffracting power of macromolecular crystals is observed and accounted for through consideration of a multi-state sequential model.
A departure from a linear or an exponential intensity decay in the diffracting power of protein crystals as a function of absorbed dose is reported. The observation of a lag phase raises the possibility of collecting significantly more data from crystals held at room temperature before an intolerable intensity decay is reached. A simple model accounting for the form of the intensity decay is reintroduced and is applied for the first time to high frame-rate room-temperature data collection.
doi:10.1107/S1399004714005379
PMCID: PMC4014120  PMID: 24816094
radiation damage; room temperature; macromolecular crystallography; dose rate
4.  Antigenic Switching of Hepatitis B Virus by Alternative Dimerization of the Capsid Protein 
SUMMARY
Chronic Hepatitis B virus (HBV) infection afflicts millions worldwide with cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a non-particulate variant of the protein (core antigen, HBcAg) that forms the building-blocks of capsids. HBeAg is not required for virion production, but is implicated in establishing immune tolerance and chronic infection. Here, we report the crystal structure of HBeAg, which clarifies how the short N-terminal propeptide of HBeAg induces a radically altered mode of dimerization relative to HBcAg (~140° rotation), locked into place through f ormation of intramolecular disulfide bridges. This structural switch precludes capsid assembly and engenders a distinct antigenic repertoire, explaining why the two antigens are cross-reactive at the T-cell level (through sequence identity) but not at the B-cell level (through conformation). The structure offers insight into how HBeAg may establish immune tolerance for HBcAg while evading its robust immunogenicity.
doi:10.1016/j.str.2012.10.017
PMCID: PMC3544974  PMID: 23219881
5.  From lows to highs: using low-resolution models to phase X-ray data 
An unusual example of how virus structure determination pushes the limits of the molecular replacement method is presented.
The study of virus structures has contributed to methodo­logical advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ∼13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.
doi:10.1107/S0907444913022336
PMCID: PMC3817700  PMID: 24189238
virus structure; phasing methods; data collection; noncrystallographic symmetry
6.  Structure of a VP1-VP3 Complex Suggests How Birnaviruses Package the VP1 Polymerase 
Journal of Virology  2013;87(6):3229-3236.
Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae, infects young salmon, with a severe impact on the commercial sea farming industry. Of the five mature proteins encoded by the IPNV genome, the multifunctional VP3 has an essential role in morphogenesis; interacting with the capsid protein VP2, the viral double-stranded RNA (dsRNA) genome and the RNA-dependent RNA polymerase VP1. Here we investigate one of these VP3 functions and present the crystal structure of the C-terminal 12 residues of VP3 bound to the VP1 polymerase. This interaction, visualized for the first time, reveals the precise molecular determinants used by VP3 to bind the polymerase. Competition binding studies confirm that this region of VP3 is necessary and sufficient for VP1 binding, while biochemical experiments show that VP3 attachment has no effect on polymerase activity. These results indicate how VP3 recruits the polymerase into birnavirus capsids during morphogenesis.
doi:10.1128/JVI.02939-12
PMCID: PMC3592137  PMID: 23283942
7.  Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution 
Nucleic Acids Research  2013;41(20):9396-9410.
Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, ɸ12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from ɸ6, ɸ8 and ɸ13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in ɸ8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in ɸ12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the ɸ12 enzyme.
doi:10.1093/nar/gkt713
PMCID: PMC3814363  PMID: 23939620
8.  Plate Tectonics of Virus Shell Assembly and Reorganization in Phage Φ8, a Distant Relative of Mammalian Reoviruses 
Structure(London, England:1993)  2013;21(8):1384-1395.
Summary
The hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity.
Graphical Abstract
Highlights
•Crystal structure of the major capsid protein P1 of the Pseudomonas phage Φ8•Φ8 P1 shares a common ancestor with mammalian reoviruses•Φ8 P1’s trapezoidal shape may facilitate capsid expansion during maturation•The pentameric organization of Φ8 P1 suggests a revised assembly pathway
El Omari et al. report a structure of the dsRNA bacteriophage ϕ8 capsid protein P1. P1 crystallizes as a pentamer, suggesting a new pathway for procapsid assembly. P1 displays a novel fold and a trapezoidal shape, distinct from that of other dsRNA virus, which may facilitate capsid expansion during maturation.
doi:10.1016/j.str.2013.06.017
PMCID: PMC3737474  PMID: 23891291
9.  Bacteriophage P23-77 Capsid Protein Structures Reveal the Archetype of an Ancient Branch from a Major Virus Lineage 
Structure(London, England:1993)  2013;21(5):718-726.
Summary
It has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. However, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. Bacteriophage P23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. We have solved multiple crystal structures of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. They fit the 14 Å resolution cryo-electron microscopy reconstruction of the entire virus exquisitely well, allowing us to propose a model for both the capsid architecture and viral assembly, quite different from previously published models. The structures of the capsid proteins and their mode of association to form the viral capsid suggest that the P23-77-like and adeno-PRD1 lineages of viruses share an extremely ancient common ancestor.
Highlights
•High-resolution structures of the two major capsid proteins of bacteriophage P23-77•P23-77 capsid proteins exhibit a conserved single β-barrel core fold•P23-77 is an ancient relative of the double β-barrel lineage of viruses•Capsid model illustrates that P23-77 uses a novel method of organization
Rissanen et al. propose a model for the architecture and assembly of bacteriophage P23-77 quite different from those previously published. The capsid proteins and their mode of association to form the virus particle suggest that P23-77 share a common evolutionary origin with the PRD1/Adenovirus lineage.
doi:10.1016/j.str.2013.02.026
PMCID: PMC3919167  PMID: 23623731
10.  Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe, Protective Vaccine Antigen 
PLoS Pathogens  2013;9(3):e1003255.
Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.
Author Summary
Picornaviruses are small RNA viruses, responsible for important human and animal diseases for example polio, some forms of the common cold and foot-and-mouth disease. Safe and effective picornavirus vaccines could in principle be produced from recombinant virus-like particles, which lack the viral genome and so cannot propagate. However the synthesis of stable forms of such particles at scale has proved very difficult. Two key problems have been that a protease required for the proper processing of the polyprotein precursor is toxic for host cells and the empty recombinant particles tend to be physically unstable in comparison to virus particles containing nucleic acid. This is particularly true in the case of Foot-and-Mouth Disease Virus (FMDV). Here we report the production and evaluation of a novel vaccine against FMDV that addresses both of these shortcomings. Importantly, the strategies we have devised to produce improved FMDV vaccines can be directly applied to viruses pathogenic for humans.
doi:10.1371/journal.ppat.1003255
PMCID: PMC3609824  PMID: 23544011
11.  Structure of a Pestivirus Envelope Glycoprotein E2 Clarifies Its Role in Cell Entry 
Cell Reports  2013;3(1):30-35.
Summary
Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.
Graphical Abstract
Highlights
► Structure of the major antigenically dominant protein of BVDV ► The overall fold of BVDV E2 shows no similarity to the class II fusion proteins ► At low pH, BVDV E2 N-terminal domain is disordered ► Entry mechanism of BVDV is probably applicable to hepatitis C virus
Stuart and colleagues have determined the atomic structure of the ectodomain of bovine viral diarrhea virus E2 glycoprotein, the major, antigenically dominant protein on the virus surface. The structure was expected to resemble the fusion molecules found on the surface of viruses such as dengue virus, but it is unlike anything previously seen. E2 itself is not, in fact, the fusion protein but binds the cell receptor and directs fusion via a pH-dependent conformational switch.
doi:10.1016/j.celrep.2012.12.001
PMCID: PMC3607223  PMID: 23273918
12.  Dame Louise Napier Johnson (1940–2012) 
Obituary for Dame Louise Napier Johnson.
doi:10.1107/S1744309112044132
PMCID: PMC3515394
13.  A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71 
Enterovirus 71 (EV71), a major agent of hand-foot-and-mouth disease in children, can cause severe central nervous system disease and mortality. At present no vaccine or antiviral therapy is available. We have determined high-resolution structures for the mature virus and natural empty particles. The structure of the mature virus is similar to that of other enteroviruses, whilst the empty particles are dramatically expanded, with notable fissures, resembling elusive enterovirus uncoating intermediates not previously characterized in atomic detail. Hydrophobic capsid pockets within the EV71 capsid are collapsed in this expanded particle, providing a detailed explanation of the mechanism for receptor-binding triggered virus uncoating. The results provide a paradigm for enterovirus uncoating, in which the VP1 GH loop acts as an adaptor-sensor for the attachment of cellular receptors, converting heterologous inputs to a generic uncoating mechanism, spotlighting novel points for therapeutic intervention.
doi:10.1038/nsmb.2255
PMCID: PMC3378640  PMID: 22388738
14.  A plate-based high-throughput assay for virus stability and vaccine formulation 
Journal of Virological Methods  2012;185(1):166-170.
Highlights
► A plate-based assay for virus measuring virus stability. ► Two fluorescent dyes measure independently but simultaneously capsid stability and capsid protein stability. ► A fast and efficient high-throughput method to optimise vaccine formulation. ► Facilitates the dissection of virus uncoating.
Standard methods for assessing the thermal stability of viruses can be time consuming and rather qualitative yet such data is a necessary requisite for vaccine formulation. In this study a novel plate-based thermal scanning assay for virus particle stability has been developed (PaSTRy: Particle Stability Thermal Release Assay). Two environment-sensitive fluorescent dyes, with non-overlapping emission spectra and different affinities, are used to accrue simultaneously independent data for the overall stability of the virus capsid, as judged by the exposure of the genome, and for capsid protein stability according to the exposure of hydrophobic side chains which are normally buried. This offers a fast and efficient high-throughput method to optimise vaccine formulation and to investigate the processes of virus uncoating.
doi:10.1016/j.jviromet.2012.06.014
PMCID: PMC3470038  PMID: 22744000
High-throughput; Virus stability; Vaccine formulation
15.  Noncatalytic Ions Direct the RNA-Dependent RNA Polymerase of Bacterial Double-Stranded RNA Virus ϕ6 from De Novo Initiation to Elongation 
Journal of Virology  2012;86(5):2837-2849.
RNA-dependent RNA polymerases (RdRps) are key to the replication of RNA viruses. A common divalent cation binding site, distinct from the positions of catalytic ions, has been identified in many viral RdRps. We have applied biochemical, biophysical, and structural approaches to show how the RdRp from bacteriophage ϕ6 uses the bound noncatalytic Mn2+ to facilitate the displacement of the C-terminal domain during the transition from initiation to elongation. We find that this displacement releases the noncatalytic Mn2+, which must be replaced for elongation to occur. By inserting a dysfunctional Mg2+ at this site, we captured two nucleoside triphosphates within the active site in the absence of Watson-Crick base pairing with template and mapped movements of divalent cations during preinitiation. These structures refine the pathway from preinitiation through initiation to elongation for the RNA-dependent RNA polymerization reaction, explain the role of the noncatalytic divalent cation in ϕ6 RdRp, and pinpoint the previously unresolved Mn2+-dependent step in replication.
doi:10.1128/JVI.05168-11
PMCID: PMC3302264  PMID: 22205747
16.  Crystallization and preliminary crystallographic analysis of the major capsid proteins VP16 and VP17 of bacteriophage P23-77 
The major capsid proteins VP16 and VP17 of bacteriophage P23-77 have been crystallized using both recombinant and purified virus and preliminary diffraction analyses have been performed.
Members of the diverse double-β-barrel lineage of viruses are identified by the conserved structure of their major coat protein. New members of this lineage have been discovered based on structural analysis and we are interested in identifying relatives that utilize unusual versions of the double-β-barrel fold. One candidate for such studies is P23-77, an icosahedral dsDNA bacteriophage that infects the extremophile Thermus thermophilus. P23-77 has two major coat proteins, namely VP16 and VP17, of a size consistent with a single-β-barrel core fold. These previously unstudied proteins have now been successfully expressed as recombinant proteins, purified and crystallized using hanging-drop and sitting-drop vapour-diffusion methods. Crystals of coat proteins VP16 and VP17 have been obtained as well as of a putative complex. In addition, virus-derived material has been crystallized. Diffraction data have been collected to beyond 3 Å resolution for five crystal types and structure determinations are in progress.
doi:10.1107/S1744309112010330
PMCID: PMC3374517  PMID: 22691792
bacteriophages; capsid proteins
17.  Cells under siege: Viral glycoprotein interactions at the cell surface 
Journal of Structural Biology  2011;175(2):120-126.
As obligate parasites, viruses are required to enter and replicate within their host, a process which employs many of their proteins to hijack natural cellular processes. High resolution X-ray crystallographic analysis has proven to be an ideal method to visualize the mechanisms by which such virus-host interactions occur and has revealed the innovative capacity of viruses to adapt efficiently to their hosts. In this review, we draw upon recently elucidated paramyxovirus-, arenavirus-, and poxvirus-host protein complex crystal structures to reveal both the capacity of viruses to appropriate one component of a physiological protein–protein binding event (often modifying it to out-compete the host-protein), and the ability to utilize novel binding sites on host cell surface receptors. The structures discussed shed light on a number of biological processes ranging from viral entry to virulence and host antagonism. Drawn together they reveal the common strategies which viruses have evolved to interact with their natural host. The structures also support molecular level rationales for how viruses can be transmitted to unrelated organisms and thus pose severe health risks.
doi:10.1016/j.jsb.2011.03.016
PMCID: PMC3137789  PMID: 21440638
Glycoprotein structure; Virus entry; Cell signaling; X-ray crystallography; Cell surface receptors; GAP, GTPase-activating protein; IPT, Ig-like plexins and transcription factors; HeV, Hendra virus; HeV-G, Hendra virus attachment glycoprotein; HNV, Henipavirus; HNV-G, Henipavirus attachment glycoprotein; NiV, Nipah virus; NiV-G, Nipah virus attachment glycoprotein; MACV, Machupo virus; PDB, protein databank; PSI, plexin-semaphrorin-integrin domain; r.m.s.d., root mean square deviation; Tf, transferrin; TfR1, transferrin receptor 1; SLAM, Signaling Lymphocytic Activation Molecule; SPINE, Structural Proteomics In Europe
18.  Automation of large scale transient protein expression in mammalian cells 
Journal of Structural Biology  2011;175(2-2):209-215.
Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI− cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative.
doi:10.1016/j.jsb.2011.04.017
PMCID: PMC3477309  PMID: 21571074
Automated tissue culture; Eukaryotic expression system; HEK 293 cells; Transient transfection; HYPERFlask
19.  How vaccinia virus has evolved to subvert the host immune response 
Journal of Structural Biology  2011;175(2-2):127-134.
Viruses are obligate intracellular parasites and are some of the most rapidly evolving and diverse pathogens encountered by the host immune system. Large complicated viruses, such as poxviruses, have evolved a plethora of proteins to disrupt host immune signalling in their battle against immune surveillance. Recent X-ray crystallographic analysis of these viral immunomodulators has helped form an emerging picture of the molecular details of virus-host interactions. In this review we consider some of these immune evasion strategies as they apply to poxviruses, from a structural perspective, with specific examples from the European SPINE2-Complexes initiative. Structures of poxvirus immunomodulators reveal the capacity of viruses to mimic and compete against the host immune system, using a diverse range of structural folds that are unique or acquired from their hosts with both enhanced and unexpectedly divergent functions.
doi:10.1016/j.jsb.2011.03.010
PMCID: PMC3477310  PMID: 21419849
Bcl-2, B-cell lymphoma-2; CPXV, Cowpox virus; dsDNA, double-stranded DNA; ECTV, ectromelia virus; GAGs, glycosaminoglycans; GPCRs, G-protein coupled receptors; IFN, interferon; IG, immunoglobulin; PDB, protein data bank; RPXV, rabbitpox virus; r.m.s.d., root mean square deviation; SPINE, Structural Proteomics In Europe; TLR, Toll-like receptors; TNF, tumour necrosis factor; TNFR, tumour necrosis factor receptor; VACV, vaccinia virus; vCCI, viral CC-chemokine inhibitor; eIF2α, eukaryotic translation initiation factor 2 alpha; TRAF6, TNF-receptor-associated factor 6; IRAKs, IL-1 receptor associated kinases; IKK, IκB kinase; Structural virology; Innate immunity; Cell signalling; X-ray crystallography; Surface receptors
20.  Recording information on protein complexes in an information management system 
Journal of Structural Biology  2011;175(2-2):224-229.
The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described.
doi:10.1016/j.jsb.2011.05.009
PMCID: PMC3477311  PMID: 21605682
Laboratory Information Management System (LIMS); Informatics; Data management; Data model; Protein complex
21.  xtalPiMS: A PiMS-based web application for the management and monitoring of crystallization trials 
Journal of Structural Biology  2011;175(2-2):230-235.
A major advance in protein structure determination has been the advent of nanolitre-scale crystallization and (in a high-throughput environment) the development of robotic systems for storing and imaging crystallization trials. Most of these trials are carried out in 96-well (or higher density) plates and managing them is a significant information management challenge. We describe xtalPiMS, a web-based application for the management and monitoring of crystallization trials. xtalPiMS has a user-interface layer based on the standards of the Protein Information Management System (PiMS) and a database layer which links the crystallization trial images to the meta-data associated with a particular crystallization trial. The user interface has been optimized for the efficient monitoring of high-throughput environments with three different automated imagers and work to support a fourth imager is in progress, but it can even be of use without robotics. The database can either be a PiMS database or a legacy database for which a suitable mapping layer has been developed.
doi:10.1016/j.jsb.2011.05.008
PMCID: PMC3477317  PMID: 21605683
Laboratory Information Management Systems (LIMS); Protein crystallization; Robotic imagers; Java web application; Data management and databases
22.  From SPINE to SPINE-2 complexes and beyond 
Journal of Structural Biology  2011;175(2-2):105-229.
doi:10.1016/j.jsb.2011.05.013
PMCID: PMC3477320  PMID: 21619933
23.  Insights into the Evolution of a Complex Virus from the Crystal Structure of Vaccinia Virus D13 
Structure(London, England:1993)  2011;19(7-12):1011-1020.
Summary
The morphogenesis of poxviruses such as vaccinia virus (VACV) sees the virion shape mature from spherical to brick-shaped. Trimeric capsomers of the VACV D13 protein form a transitory, stabilizing lattice on the surface of the initial spherical immature virus particle. The crystal structure of D13 reveals that this major scaffolding protein comprises a double β barrel “jelly-roll” subunit arranged as pseudo-hexagonal trimers. These structural features are characteristic of the major capsid proteins of a lineage of large icosahedral double-stranded DNA viruses including human adenovirus and the bacteriophages PRD1 and PM2. Structure-based phylogenetic analysis confirms that VACV belongs to this lineage, suggesting that (analogously to higher organism embryogenesis) early poxvirus morphogenesis reflects their evolution from a lineage of viruses sharing a common icosahedral ancestor.
Highlights
► Poxvirus D13 acts as a scaffold for the morphogenesis of spherical immature virions ► D13 has a double “jelly-roll” structure, like other large DNA virus capsid proteins ► Structure-based phylogenetics places D13 into an icosahedral viral lineage ► Poxvirus morphogenesis reflects Vaccinia virus evolution from an icosahedral ancestor
doi:10.1016/j.str.2011.03.023
PMCID: PMC3136756  PMID: 21742267
24.  Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells 
The α-mannosidase I inhibitor kifunensine inhibited N-glycan processing in long-term cultures of Chinese hamster ovary cells, allowing deglycosylation and crystallization of the homodimeric extracellular region of the inhibitory glycoprotein receptor CTLA-4 (CD152).
Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3–5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3–4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a = 43.9, b = 51.5, c = 102.9 Å and space group P212121 that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems.
doi:10.1107/S1744309111017672
PMCID: PMC3144796  PMID: 21795794
kifunensine; glycoproteins; cytotoxic T-lymphocyte antigen 4
25.  Crystallization and preliminary X-ray analysis of mouse RANK and its complex with RANKL 
Mouse RANKL and its receptor RANK have been cloned, expressed and purified. Crystals of RANK alone and in complex with RANKL have been obtained from which diffraction data have been collected to 2.0 and 2.8 Å resolution, respectively.
The interaction between the TNF-family molecule receptor activator of NF-κB ligand (RANKL) and its receptor RANK induces osteoclast formation, activation and survival in the process of bone remodelling. RANKL–RANK also plays critical roles in T-cell/dendritic cell communication and lymph-node formation and in a variety of pathologic conditions such as tumour-cell migration and bone metastasis. Both the ectodomain of mouse RANKL and the extracellular domain of mouse RANK have been cloned, expressed and purified. Crystals of RANK alone and of RANK in complex with RANKL have been obtained that are suitable for structure determination.
doi:10.1107/S1744309109015735
PMCID: PMC2688419  PMID: 19478440
RANK; RANKL; OPG; RANKL–RANK complex; TNF superfamily

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