EMBL Outstation Research Articles

Ending the “Publish and Vanish” Culture: How the Data Standardization Process Will Assist in Data Harvesting

ADVERTISEMENT RETURN TO ISSUEEditorialNEXTEnding the “Publish and Vanish” Culture: How the Data Standardization Process Will Assist in Data HarvestingSandra OrchardView Author Information EMBL Outstation - European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, U.K.Cite this: J. Proteome Res. 2009, 8, 7, 3219Publication Date (Web):July 6, 2009Publication History Published online6 July 2009Published inissue 6 July 2009https://doi.org/10.1021/pr900527rCopyright © 2009 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views577Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (107 KB) Get e-AlertsSUBJECTS:Biological databases,Deposition,Protein dynamics,Proteomics Get e-Alerts

C1 Inhibitor Serpin Domain Structure Reveals the Likely Mechanism of Heparin Potentiation and Conformational Disease

C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded beta-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack.

Structural Organization of Essential Iron-Sulfur Clusters in the Evolutionarily Highly Conserved ATP-binding Cassette Protein ABCE1

The ABC protein ABCE1, formerly named RNase L inhibitor RLI1, is one of the most conserved proteins in evolution and is expressed in all organisms except eubacteria. Because of its fundamental role in translation initiation and/or ribosome biosynthesis, ABCE1 is essential for life. Its molecular mechanism has, however, not been elucidated. In addition to two ABC ATPase domains, ABCE1 contains a unique N-terminal region with eight conserved cysteines, predicted to coordinate iron-sulfur clusters. Here we present detailed information on the type and on the structural organization of the Fe-S clusters in ABCE1. Based on biophysical, biochemical, and yeast genetic analyses, ABCE1 harbors two essential diamagnetic [4Fe-4S](2+) clusters with different electronic environments, one ferredoxin-like (CPX(n)CX(2)CX(2)C; Cys at positions 4-7) and one unique ABCE1-type cluster (CXPX(2)CX(3)CX(n)CP; Cys at positions 1, 2, 3, and 8). Strikingly, only seven of the eight conserved cysteines coordinating the Fe-S clusters are essential for cell viability. Mutagenesis of the cysteine at position 6 yielded a functional ABCE1 with the ferredoxin-like Fe-S cluster in a paramagnetic [3Fe-4S](+) state. Notably, a lethal mutation of the cysteine at position 4 can be rescued by ligand swapping with an adjacent, extra cysteine conserved among all eukaryotes.

The Iron-Sulfur Cluster-free Hydrogenase (Hmd) Is a Metalloenzyme with a Novel Iron Binding Motif

The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing cofactor of yet unknown structure. X-ray absorption spectroscopy of the active, as isolated enzyme from Methanothermobacter marburgensis (mHmd) and of the active, reconstituted enzyme from Methanocaldococcus jannaschii (jHmd) revealed the presence of mononuclear iron with two CO, one sulfur and one or two N/O in coordination distance. In jHmd, the single sulfur ligand is most probably provided by Cys176, as deduced from a comparison of the activity and of the x-ray absorption and Mössbauer spectra of the enzyme mutated in any of the three conserved cysteines. In the isolated Hmd cofactor, two CO, one sulfur, and two nitrogen/oxygen atoms coordinate the iron, the sulfur ligand being most probably provided by mercaptoethanol, which is absolutely required for the extraction of the iron-containing cofactor from the holoenzyme and for the stabilization of the extracted cofactor. In active mHmd holoenzyme, the number of iron ligands increased by one when one of the Hmd inhibitors (CO or KCN) were present, indicating that in active Hmd, the iron contains an open coordination site, which is proposed to be the site of H2 interaction.

Structural Studies on a Mitochondrial Glyoxalase II

Glyoxalase 2 is a beta-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and (1)H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 +/- 0.15 eq of iron and 1.31 +/- 0.05 eq of Zn(II) and to exhibit k(cat) and K(m) values of 129 +/- 10 s(-1) and 391 +/- 48 microm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. (1)H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-A resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).

Calf spleen purine-nucleoside phosphorylase: crystal structure of the binary complex with a potent multisubstrate analogue inhibitor.

Purine-nucleoside phosphorylase (PNP) deficiency in humans leads to inhibition of the T-cell response. Potent membrane-permeable inhibitors of this enzyme are therefore considered to be potential immunosuppressive agents. The binary complex of the trimeric calf spleen phosphorylase, which is highly homologous to human PNP, with the potent ground-state analogue inhibitor 9-(5,5-difluoro-5-phosphonopentyl)guanine (DFPP-G) was crystallized in the cubic space group P2(1)3, with unit-cell parameter a = 93.183 A and one monomer per asymmetric unit. High-resolution X-ray diffraction data were collected using synchrotron radiation (EMBL Outstation, DESY, Hamburg, station X13). The crystal structure was refined to a resolution of 2.2 A and R and Rfree values of 19.1 and 24.2%, respectively. The crystal structure confirms that DFPP-G acts as a multisubstrate analogue inhibitor as it binds to both nucleoside- and phosphate-binding sites. The structure also provides the answers to some questions regarding the substrate specificity and molecular mechanism of trimeric PNPs. The wide access to the active-site pocket that was observed in the reported structure as a result of the flexibility or disorder of two loops (residues 60-65 and 251-266) strongly supports the random binding of substrates. The putative hydrogen bonds identified in the base-binding site indicate that N1-H and not O6 of the purine base defines the specificity of trimeric PNPs. This is confirmed by the fact that the contact of guanine O6 with Asn243 Odelta1 is not a direct contact but is mediated by a water molecule. Participation of Arg84 in the binding of the phosphonate group experimentally verifies the previous suggestion [Blackburn & Kent (1986), J. Chem. Soc. Perkin Trans. I, pp. 913-917; Halazy et al. (1991), J. Am. Chem. Soc. 113, 315-317] that fluorination of alkylphosphonates yields compounds with properties that suitably resemble those of phosphate esters and in turn leads to optimized interactions of such analogues with the phosphate-binding site residues. DFPP-G shows a Ki(app) in the nanomolar range towards calf and human PNPs. To date, no high-resolution X-ray structures of these enzymes with such potent ground-state analogue inhibitors have been available in the Protein Data Bank. The present structure may thus be used in the rational structure-based design of new PNP inhibitors with potential medical applications.

Expression, purification and crystallization of two peroxisomal acyl-CoA oxidases from Arabidopsis thaliana.

Two members of the acyl-CoA oxidase family from Arabidopsis thaliana have been cloned, overexpressed, purified and crystallized. Long-chain-specific acyl-CoA oxidase 1 crystals are characterized by a large variation in diffraction quality and non-isomorphous unit-cell parameters. The best crystals diffract to 2.0 angstrom using synchrotron radiation, have unit-cell parameters a = 85.2, b = 117.0, c = 131.0 angstrom, alpha = beta = gamma = 90 degrees and show P2(1)2(1)2(1) symmetry. There are two polypeptide chains in the asymmetric unit. Short-chain-specific acyl-CoA oxidase 4 crystals are trigonal, space group P3(1)21/P3(2)21, with unit-cell parameters a = b = 198.7, c = 149.6 angstroms. The crystals are most likely to contain six polypeptide chains in the asymmetric unit. Freshly prepared acyl-CoA oxidase 4 crystals diffract to 3.9 angstroms at cryogenic temperature at beamline I711, Max-Lab, but the diffraction quality degenerates after storage for only a few days in the crystallization drop. A selenomethionine-substituted form of the protein was produced and two-wavelength MAD data were collected at beamline BW7A, EMBL Outstation, Hamburg.

Calf spleen purine nucleoside phosphorylase: structure of its ternary complex with an N(7)-acycloguanosine inhibitor and a phosphate anion.

The calf spleen purine nucleoside phosphorylase (PNP) ternary complex with an N(7)-acycloguanosine inhibitor and a phosphate ion has been crystallized in the cubic space group P2(1)3, with unit-cell parameter a = 94.11 A and one monomer per asymmetric unit. X-ray diffraction data were collected using synchrotron radiation (Station X31, EMBL Outstation, DESY, Hamburg). The crystal structure was refined to a resolution of 2.2 A and R and R(free) values of 17.5 and 24.5%, respectively. The acyclonucleoside inhibitor is bound in the active site in an inverted ('upside-down') orientation of the purine base compared with natural substrates. The side chain of Asp243 forms two hydrogen bonds with the base ring: N(delta) donates a hydrogen to N(3) and O(delta) accepts a hydrogen from the guanine N(2)-amino group. N(1)--H of the base is hydrogen bonded to O(epsilon) of Glu201, while N(9) accepts a hydrogen bond from Thr242 O(gamma). In addition, a water molecule (W417) bridges the N(2)-amino group of the base and O(epsilon) of Glu201. In the phosphate-binding site, a phosphate ion is bound to Ser33, His64, Arg84, His86, Ala116 and Ser220. The acyclic chain of the N(7)-acycloguanosine inhibitor is in a folded conformation and together with a water molecule (W388) occupies the pentose-binding site, with possible hydrogen bonds to Tyr88 O(eta) and His257 N(delta 1). This new binding mode fully accounts for the previously observed substrate properties of 7-beta-D-ribofuranosides of hypoxanthine and guanine. It also provides a new starting point for the design of inhibitors of PNP for therapeutic and other applications.

Resources at EBI.

AbstractThe European Bioinformatics Institute (EBI) is an EMBL Outstation located at the Wellcome Trust Genome Campus in Hinxton (UK). The EBI maintains and distributes the EMBL Nucleotide Sequence database, Europe’s primary nucleotide sequence data resource, the SwissProt Protein Sequence database, in collaboration with Amos Bairoch of the Swiss Institute for Bioinformatics (SIB), TrEMBL a SwissProt complement consisting of translations from EMBL database coding sequences, MSD, the Molecular Structure Database in collaboration with the Protein Data Bank (PDB) in Brookhaven National Laboratory (Brookhaven, NY), the Radiation Hybrid database (RHdb), and other additional molecular biology databases produced in collaboration with other groups. The EBI also provides network services that allow access to the most up-to-date data collections via the Internet through its World Wide Web interfaces and ftp services, also providing database and sequence similarity searches facilities.KeywordsProtein Data BankEuropean Bioinformatics InstituteEMBL DatabaseData SubmissionGenome Survey SequenceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

RHScoresFormat: a tool for formatting radiation hybrid data

Genome maps are important tools in genetic research. They also provide frameworks for large-scale sequencing. A variety of methods have been developed to generate maps from experimental data (Refs [1xCox, D.R. et al. Science. 1990; 250: 245–250Crossref | PubMedSee all References, 2xBoehnke, M., Lange, K., and Cox, D.R. Am. J. Hum. Genet. 1991; 49: 1174–1188PubMedSee all References]). A database for collecting and distributing radiation hybrid raw data (the `scores' representing the retention patterns of a given STS assayed on a specific panel) has been maintained at the European Bioinformatics Institute (EMBL Outstation, Hinxton) since 1995 (Ref. [3xLijnzaad, P., Helgesen, C., and Rodriguez-Tome, P. Nucleic Acids Res. 1998; 26: 102–105Crossref | PubMed | Scopus (16)See all References][3]). This database is available at URL http://www.ebi.ac.uk/RHdbVarious analytical methods and software for constructing radiation hybrid maps have been developed in recent years. Unfortunately, each of these programs requires a different input format for the data. We have developed the RHScoresFormat tool to translate the user data given in a simple format to the format used by a specific program. Translators for the most common mapping programs have been implemented. These include: RHMAP (Ref. [2xBoehnke, M., Lange, K., and Cox, D.R. Am. J. Hum. Genet. 1991; 49: 1174–1188PubMedSee all References][2]), RHMAPPER and RHMapper server [L. Stein, L. Kruglyak, D. Slonim and E. Lander (1995) RHMAPPER, unpublished software, Whitehead Institute/MIT Center for Genome Research], RADMAP/ MULTIMAP (Refs [4xMatise, T., Perlin, M., and Chakravarti, A. Nat. Genet. 1994; 6: 384–390Crossref | PubMed | Scopus (376)See all References, 5xMatise, T.C. and Chakravarti, A. Am. J. Hum. Genet. 1995; 57: A15See all References]), and Stanford RH Server (http://www.shgc.stanford.edu/ RH).It is also possible to query the Radiation Hybrid database through a set of query windows by: STS name, chromosome, RHdb release, species, etc. The results can be merged with the user's own data to be used conjointly in the analysis.The RHScoresFormat Applet is available on the web at: http://corba.ebi.ac.uk/RHdb/Clients, which links also to all the other RHdb clients. A browser supporting Java (minimum JDK1.1.4) is needed to run the applets. The application is also available from the EBI ftp anonymous server (ftp://ftp.ebi.ac.uk/pub/databases/RHdb/software).

How X-ray Diffraction with Synchrotron Radiation Got Started.

The need to record low-angle-scattering X-ray fibre diagrams from muscle with millisecond time resolution drove the use of synchrotron radiation as an X-ray light source. The first smudgy diffraction patterns were obtained from a slice of insect flight muscle. Out of this grew the EMBL Outstation at DESY.

Development of Synchrotron Radiation as a High-Intensity Source for X-ray Diffraction

Interest in the molecular mechanism of muscle contraction led to the search for an intense source of X-rays of 1-2 A wavelength so as to be able to examine the rich X-ray diffraction patterns given by muscles during contraction. This led to the first X-ray diffraction experiments using synchrotron radiation, carried out by Holmes, Rosenbaum and Witz at DESY, Hamburg, in September 1970. In the following years, the EMBL Outstation, to utilize synchrotron radiation for biological structure determination, was established at DESY and preliminary experiments on muscle were also carried out at NINA (Daresbury). The development of time-resolved techniques for muscle diffraction was first started in the MRC Molecular Biology Laboratory in Cambridge, using rotating-anode X-ray tubes, and was then greatly extended at the EMBL Outstation, Hamburg, using the storage ring DORIS. This was a very successful venture, and helped to drive the whole technology development and to interest other potential users in the technique.

Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with hypoxanthine at 2.15 Å resolution

Trimeric calf spleen purine nucleoside phosphorylase has been complexed with hypoxanthine via phosphorolysis of inosine in the presence of phosphate. The resulting “Michaelis” complex (three hypoxanthine molecules per trimer), presumed to be formed under these conditions, crystallized in the cubic space group P2 13, with unit cell dimension a = 94.11 Å and one monomer in the asymmetric crystal unit; the biologically active trimer is located on the crystallographic 3-fold axis. High-resolution X-ray diffraction data were collected using synchrotron radiation (EMBL outstation, Hamburg, c/o DESY). The crystal structure has been determined by molecular replacement and refined at 2.15 Å resolution to an R-value of 0.18. In the hypoxanthine binding site, a cis-peptide bond between Asn243 and Lys244 is observed. Side-chains of Glu201 and Asn243, as well as one integral water molecule located in the base binding site, form hydrogen bonds with the hypoxanthine N-1 H, N-7 H and O-6. A second water molecule links the base positions N-3 and N-9 with an adjacent pocket, which presumably is the phosphate-binding site. This pocket is filled completely by a cluster of six water molecules. Hence all possible donor/acceptor-positions of hypoxanthine are saturated by hydrogen- bonding to protein side-chains or integral water molecules. Purine nucleoside phosphorylase isolated form human tissues is a primary target for chemotherapeutic intervention, and the more stable calf enzyme has similar physico-chemical and kinetic properties, as well as response to inhibitors. Hence the high-resolution structure presented here may serve for design of inhibitors with potential pharmacological applications.

X-ray instrumentation for a focused wiggler beamline at the EMBL Outstation Hamburg

A flexible setup consisting of two independent water-cooled X-ray monochromators providing radiation for two separate beamlines (A and B) has been installed on the multipole wiggler BW7 at the DORIS III storage ring. The first optical element of both lines is a Rh-coated SiC mirror that reduces the heat load on subsequent energy defining elements. Line B, which will be predominantly used for protein crystallography, is optimized for flux and employs a triangular monochromator and segmented glass mirror for horizontal and vertical focusing, respectively; it can be operated in a wavelength range from 0.5 to 1.8 Å. Line A features a high-energy resolution fixed exit, nondispersive double monochromator, optimized for accurate and fast energy scans. It will mainly be used for anomalous scattering, EXAFS, and small-angle scattering. The use of encoders, intelligent stepper/dc-motor controllers, and position sensitive beam monitors allow a comfortable and fast alignment of all axes. Both experimental hutches are equipped with large motorized optical tables, offering a versatile and convenient mounting surface for experiments.

Structure of the ADP complex of the 3-phosphoglycerate kinase from Bacillus stearothermophilus at 1.65 A.

The structure of the ADP complex of the enzyme 3-phosphoglycerate kinase (PGK, E.C. 2.7.2.3) from Bacillus stearothermophilus NCA-1503 has been determined by the method of molecular replacement. The structure has been refined to an R factor of 0.16 for all data between 10.0 and 1.65 A resolution, using data collected on the Hendrix-Lentfer imaging plate at the EMBL outstation in Hamburg. The r.m.s. deviations from stereochemical ideality are 0.010 and 0.011 A for bonds and planes, respectively. Although crystallized in the presence of the nucleotide product MgATP, the high-resolution structure reveals the bound nucleotide to be MgADP reflecting the low intrinsic ATPase activity of PGK. Although the two domains of this enzyme are found to be some 4.5 degrees closer together than is found in the yeast and horse-muscle apo-enzyme structures, this structure represents the 'open' rather than the 'closed', catalytically competent form, of the enzyme.

Initiating a crystallographic study of a class II fructose-1,6-bisphosphate aldolase

We have reproducibly crystallized the metal-dependent Class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of truncated hexagonal bipyramids have unit cell dimensions of a = b = 78.4 A ̊ , c = 290.6 A ̊ and are suitable for a detailed structural analysis. The space group has been identified as P6 122 or enantiomorph. Data sets to approximately 2.9 Å resolution have been recorded using both the Rigaku R-AXIS IIc image plate area detector coupled to a copper target rotating anode X-ray source and using the MAR image plate systems with synchrotron radiation at the EMBL outstation DESY in Hamburg, and at S.R.S. Daresbury. Diffraction beyond 2.5 Å has been observed when large freshly grown crystals are used with the synchrotron beam. A data set to this resolution has been collected. Several putative heavy-atom derivative data sets have also been measured using synchrotron radiation facilities and analysis of these data sets is in progress.

The EMBL-Hamburg imaging plate scanner (abstract)

At the EMBL outstation in Hamburg, imaging plate scanners have now been operational for the last two years. The success can be called enormous, despite the disadvantage of a ″very slow″ read-out time, which is after all not longer than for scientific charge coupled devices. We will discuss the principles of operation of our scanner and illustrate its characteristics by measured results. Some important features of the Hamburg scanner are (i) From its characteristics it can be said that this system combines an excellent spatial resolution with the high sensitivity of a single photon counter, and negligible intrinsic noise. (ii) The virtually constant sensitivity at short wavelengths, down to 0.5 Å, makes this detection system ideal for protein work. The lack of absorption reduces radiation damage to a minimum and makes absorption corrections unnecessary. (iii) The exposure times are reduced by typically a factor of 20–50 as compared to photographic film. (iv) These short exposure times demand for the shortest possible read-out times. The handling of plates at a synchrotron source would become quite cumbersome. Our scanner has been optimized for a minimum read-out time without spoiling the accuracy of the data. (v) A single computer collects, stores, displays, and processes the crystallographic data. (vi) Its reliability and insensitivity to environmental changes (i.e., temperature) make it an ideal device for long term automatic data collection with only a minimum of servicing required. (vii) The spatial resolution (150 μm×150 μm) does not represent the ultimate intrinsic limit of the imaging plate, but is a common sense choice in order to avoid the need of excessive data storage space. (viii) The large size of the imaging plate matches the dimensions of the reflections obtained with average size crystals and x-ray beams very well. Up to 100 orders can be resolved in practice, at each side of the center of the plate. This limitation is not dictated by the spatial resolution of the scanner, but rather by the actual spot sizes in the patterns.

Data acquisition systems for linear and area X-ray detectors using delay line readout

Digital systems for fast readout of linear and area position sensitive detectors based on commercially available CAMAC modules are described. The first version of the system has been used extensively for synchrotron radiation work carried out at the EMBL outstation in Hamburg during the last five years. A second system allowing measurements at count rates up to 600 kHz on a continuous source has been recently installed. An overview of the complete acquisition and information processing system is also given.

Data appraisal, evaluation and display for synchrotron radiation experiments: Hardware and software

The hardware and software of the data appraisal, evaluation and display systems used for synchrotron radiation experiments at the EMBL Outstation on the storage ring DORIS are described.The system is based on CAMAC hardware and modular software and has been extensively used for a variety of applications in EXAFS and small angle scattering and diffraction.

Fluorescence EXAFS detection at the EMBL synchrotron radiation outstation, Hamburg: hardware, software and operational experience

A fluorescence detection system for biological EXAFS experiments has been set up at the EMBL Outstation, Hamburg. An array of ten NaI(Tl) scintillation counters is used. The total counting rate of the system is 1.4 MHz. Data collection and reduction procedures were developed and are described. Examples of spectra obtained are presented. The system makes possible studies of samples in the sub-millimolar concentration range in the most favourable spectral region.