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PubMed2001

A rapid classification protocol for the CATH Domain Database to support structural genomics.

In order to support the structural genomic initiatives, both by rapidly classifying newly determined structures and by suggesting suitable targets for structure determination, we have recently developed several new protocols for classifying structures in the CATH domain database (http://www.biochem.ucl.ac.uk/bsm/cath). These aim to increase the speed of classification of new structures using fast algorithms for structure comparison (GRATH) and to improve the sensitivity in recognising distant structural relatives by incorporating sequence information from relatives in the genomes (DomainFinder). In order to ensure the integrity of the database given the expected increase in data, the CATH Protein Family Database (CATH-PFDB), which currently includes 25,320 structural domains and a further 160,000 sequence relatives has now been installed in a relational ORACLE database. This was essential for developing more rigorous validation procedures and for allowing efficient querying of the database, particularly for genome analysis. The associated Dictionary of Homologous Superfamilies [Bray,J.E., Todd,A.E., Pearl,F.M.G., Thornton,J.M. and Orengo,C.A. (2000) Protein Eng., 13, 153-165], which provides multiple structural alignments and functional information to assist in assigning new relatives, has also been expanded recently and now includes information for 903 homologous superfamilies. In order to improve coverage of known structures, preliminary classification levels are now provided for new structures at interim stages in the classification protocol. Since a large proportion of new structures can be rapidly classified using profile-based sequence analysis [e.g. PSI-BLAST: Altschul,S.F., Madden,T.L., Schaffer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. (1997) Nucleic Acids Res., 25, 3389-3402], this provides preliminary classification for easily recognisable homologues, which in the latest release of CATH (version 1.7) represented nearly three-quarters of the non-identical structures.

Computational Biology↗
PubMed2002

Structural similarity to link sequence space: new potential superfamilies and implications for structural genomics.

The current pace of structural biology now means that protein three-dimensional structure can be known before protein function, making methods for assigning homology via structure comparison of growing importance. Previous research has suggested that sequence similarity after structure-based alignment is one of the best discriminators of homology and often functional similarity. Here, we exploit this observation, together with a merger of protein structure and sequence databases, to predict distant homologous relationships. We use the Structural Classification of Proteins (SCOP) database to link sequence alignments from the SMART and Pfam databases. We thus provide new alignments that could not be constructed easily in the absence of known three-dimensional structures. We then extend the method of Murzin (1993b) to assign statistical significance to sequence identities found after structural alignment and thus suggest the best link between diverse sequence families. We find that several distantly related protein sequence families can be linked with confidence, showing the approach to be a means for inferring homologous relationships and thus possible functions when proteins are of known structure but of unknown function. The analysis also finds several new potential superfamilies, where inspection of the associated alignments and superimpositions reveals conservation of unusual structural features or co-location of conserved amino acids and bound substrates. We discuss implications for Structural Genomics initiatives and for improvements to sequence comparison methods.

Amino Acid Sequence↗
PubMed2002

[Genomic structure of low density lipoprotein receptor related protein 5 gene].

OBJECTIVE: To determine the genomic structure of low density lipoprotein receptor related protein 5 (LRP5) gene. METHODS: cDNA sequence encoding LRP5 was used to screen genomic clones containing LRP5 gene by computer hybridization approach. By comparing the cDNA sequence of LRP5 with the genomic sequences, the genomic structure of LRP5 was determined, and then it was conformed by amplifying and sequencing the sequences of exons and splicing junction. RESULTS: The genomic sequence of LRP5 gene was 131.6 kb in length, containing 23 exons and 22 introns. Three single nucleotide polymorphisms were detected within the coding sequences of LRP5 gene, namely A459G in exon 2, C2220T in exon 10 and G4416C in exon 21. Four polymorphic markers, D11S1917, D11S4087, D11S1337 and D11S4178, located in the 5' flank sequence, introns 1, 4, and 13 of the LRP5 gene, respectively. CONCLUSION: The characterization of genomic structure of LRP5 gene allows the investigators to detect disease-causing mutation within the gene and further study the function of LRP5 gene.

Base Sequence↗
PubMed2005

Rapid isolation of single-chain antibodies for structural genomics.

High throughput approaches to structural genomics requires expression, purification, and crystallization of proteins derived from predicted open reading frames cloned into a host organism, typically E. coli. Early results from this approach suggest that the success rate of obtaining well diffracting crystals from eukaryotic proteins is disappointingly low. A proven method of improving the odds of crystallization is formation of a complex with a conformation-stabilizing partner of known structure that is easily crystallized. Such complexes are also able to engage in different crystal contacts than the original protein by itself. Fab fragments derived from monoclonal antibodies have been successfully used for this purpose for a variety of proteins, however conventional methods for the isolation of monoclonal antibodies from hybridomas are time consuming and expensive. We are exploring the use of phage display to generate recombinant antibodies to target proteins that can be used to obtain co-complexes to facilitate crystallization and structural determination. We are using a large, human single-chain Fv (scFv) library to select for antibodies that bind to a panel of Leishmania major target proteins. Thirteen out of 16 target proteins yielded good binders after three rounds of enrichment. A total of 55 distinct scFvs were identified, with five targets each yielding at least five different scFvs. Individual clones were analyzed for binding specificity and soluble scFv can be readily produced and purified via the appended His(6) epitope tag. Using immunoaffinity chromatography, eight scFv target protein pairs were identified that exhibit stable complex formation and are suitable for co-crystallization trials.

Animals↗
PubMed2005

High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics.

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.

Arabidopsis↗
PubMed2005

Prion-like Doppel gene (PRND) in the goat: genomic structure, cDNA, and polymorphisms.

The genomic structure of the caprine Doppel gene (PRND) was determined using the ovine sequence as a scaffold to generate PCR fragments that were aligned with a cDNA sequence obtained from testicular mRNA. The caprine gene contains two exons, 89 and >2291 bp long, separated by a 1689-bp intron. Two mRNA isoforms of 3.2 and 4.8 kb were identified in the testis, as well as the exact transcription start site by fluorescently labeled oligonucleotide extension (FLOE). Like in sheep and cattle, the open reading frame (ORF) (537 bp) lies within exon 2 and is very much conserved in sheep (99.3%) and cattle (97%). The intronic sequence is also highly conserved (95.3%) compared with sheep, with the only exception of a 47-bp insertion. The PRND ORF was sequenced in 47 healthy and 17 TSE-affected goats of the Italian Ionica breed. Seven nucleotide positions showed variation: T28C, C65T, A151G, G286A, C385G, T451C, and T528C. Five were commonly represented polymorphisms: T28C, T451C, and T528C are silent mutations at codons L10, L151, and I176, respectively, while A151G and C385G determine a T51A and L129V amino acid change, respectively. The two remaining variants, C65T and G286A, were rare, leading to the amino acid substitutions S22F and E96K, respectively. None of the polymorphisms was significantly relatable to the TSE status, and the same result was obtained by the analysis of the combined haplotypes at the five major polymorphic sites, namely, T28C, C65T, A151G, G286A, and C385G.

Amino Acid Sequence↗
PubMed1999

The PRESAGE database for structural genomics.

The PRESAGE database is a collaborative resource for structural genomics. It provides a database of proteins to which researchers add annotations indicating current experimental status, structural predictions and suggestions. The database is intended to enhance communication among structural genomics researchers and aid dissemination of their results. The PRESAGE database may be accessed at http://presage.stanford.edu/

Databases, Factual↗
PubMed1999

A comparison of sequence and structure protein domain families as a basis for structural genomics.

MOTIVATION: Protein families can be defined based on structure or sequence similarity. We wanted to compare two protein family databases, one based on structural and one on sequence similarity, to investigate to what extent they overlap, the similarity in definition of corresponding families, and to create a list of large protein families with unknown structure as a resource for structural genomics. We also wanted to increase the sensitivity of fold assignment by exploiting protein family HMMs. RESULTS: We compared Pfam, a protein family database based on sequence similarity, to Scop, which is based on structural similarity. We found that 70% of the Scop families exist in Pfam while 57% of the Pfam families exist in Scop. Most families that occur in both databases correspond well to each other, but in some cases they are different. Such cases highlight situations in which structure and sequence approaches differ significantly. The comparison enabled us to compile a list of the largest families that do not occur in Scop; these are suitable targets for structure prediction and determination, and may be useful to guide projects in structural genomics. It can be noted that 13 out of the 20 largest protein families without a known structure are likely transmembrane proteins. We also exploited Pfam to increase the sensitivity of detecting homologs of proteins with known structure, by comparing query sequences to Pfam HMMs that correspond to Scop families. For SWISSPROT+TREMBL, this yielded an increase in fold assignment from 31% to 42% compared to using FASTA only. This method assigned a structure to 22% of the proteins in Saccharomyces cerevisiae, 24% in Escherichia coli, and 16% in Methanococcus jannaschii.

Computational Biology↗
PubMed2003

Patenting nonassociated polymeric structures (NAPS): implications for structural genomic data release.

The intellectual property laws that govern patent rights should provide a reasonable balance between the competing concerns of open access and exclusivity. Open access can facilitate knowledge dissemination and collaboration in furthering science. On the other hand, exclusivity can ensure interest and financial investment in scientific research and development. In recent days, the appropriate balance between open access and exclusivity has been a focus of public debate, particularly with regard to genomic inventions and their applications. In seeking to reconcile the timing of structural genomic data release with certain efforts to secure intellectual property rights, the International Structural Genomics Organisation joins others confronting this controversy. This paper seeks to inform the discussion with an overview of the U.S. standards for patenting nonassociated polymeric structures (NAPS), which include polynucleotides or polypeptides of unknown biological significance, and their corresponding structural data. In the United States, the present ability to obtain patent rights to these discoveries appears problematic given the requirement of specific, substantial and credible utility, among other things. Without demonstrable utility, NAPS and NAPS-related data likely will not be entitled to patent protection, whether the U.S. Patent & Trademark Office rejects NAPS claims as unpatentable in the first instance, or the U.S. federal courts invalidate NAPS claims in later patent litigation. As such, the improbability of obtaining enforceable patent rights to NAPS might undermine the rationale for delaying structural genomic data release to allow for the filing of patent applications in this regard.

Expressed Sequence Tags↗
PubMed2003

Structure-based functional inference in structural genomics.

The dramatically increasing number of new protein sequences arising from genomics and proteomics requires the need for methods to rapidly and reliably infer the molecular and cellular functions of these proteins. One such approach, structural genomics, aims to delineate the total repertoire of protein folds in nature, thereby providing three-dimensional folding patterns for all proteins and to infer molecular functions of the proteins based on the combined information of structures and sequences. The goal of obtaining protein structures on a genomic scale has motivated the development of high throughput technologies and protocols for macromolecular structure determination that have begun to produce structures at a greater rate than previously possible. These new structures have revealed many unexpected functional inferences and evolutionary relationships that were hidden at the sequence level. Here, we present samples of structures determined at Berkeley Structural Genomics Center and collaborators' laboratories to illustrate how structural information provides and complements sequence information to deduce the functional inferences of proteins with unknown molecular functions. Two of the major premises of structural genomics are to discover a complete repertoire of protein folds in nature and to find molecular functions of the proteins whose functions are not predicted from sequence comparison alone. To achieve these objectives on a genomic scale, new methods, protocols, and technologies need to be developed by multi-institutional collaborations worldwide. As part of this effort, the Protein Structure Initiative has been launched in the United States (PSI; www.nigms.nih.gov/funding/psi.html). Although infrastructure building and technology development are still the main focus of structural genomics programs, a considerable number of protein structures have already been produced, some of them coming directly out of semiautomated structure determination pipelines. The Berkeley Structural Genomics Center (BSGC) has focused on the proteins of Mycoplasma or their homologues from other organisms as its structural genomics targets because of the minimal genome size of the Mycoplasmas as well as their relevance to human and animal pathogenicity (http://www.strgen.org). Here we present several protein examples encompassing a spectrum of functional inferences obtainable from their three-dimensional structures in five situations, where the inferences are new and testable, and are not predictable from protein sequence information alone.

Adenosine Triphosphate↗
PubMed2003

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

Amino Acid Sequence↗
PubMed2003

Secure web book to store structural genomics research data.

Recently established collaborative structural genomics programs aim at significantly accelerating the crystal structure analysis of proteins. These large-scale projects require efficient data management systems to ensure seamless collaboration between different groups of scientists working towards the same goal. Within the Berlin-based Protein Structure Factory, the synchrotron X-ray data collection and the subsequent crystal structure analysis tasks are located at BESSY, a third-generation synchrotron source. To organize file-based communication and data transfer at the BESSY site of the Protein Structure Factory, we have developed the web-based BCLIMS, the BESSY Crystallography Laboratory Information Management System. BCLIMS is a relational data management system which is powered by MySQL as the database engine and Apache HTTP as the web server. The database interface routines are written in Python programing language. The software is freely available to academic users. Here we describe the storage, retrieval and manipulation of laboratory information, mainly pertaining to the synchrotron X-ray diffraction experiments and the subsequent protein structure analysis, using BCLIMS.

Crystallography, X-Ray↗
PubMed2003

Structural genomics of lipid signaling domains.

Signaling domains have been identified by the analysis of data derived from biochemical studies, molecular cloning, or genetic studies. With the availability of genomic information from many organisms and the improved sensitivity in homology detection techniques, many new domains are being identified. In an attempt to understand biochemical and biological function of these domains, we have started a small-scale structural genomics, or structural biology with genomic approach. Two examples from our recent work are steroidogenic acute regulatory protein (StAR)-related lipid-transfer (START) domain and inositol polyphosphate 5-phosphatase catalytic (IPP5C) domain. Crystal structure of human MLN64-START domain revealed a hollowed-out protein containing a hydrophobic tunnel just large enough to bind one molecule of cholesterol and completely exclude it from solvent. This structure suggests that the START domain is a classical type of lipid transporter. On the contrary, the function of IPP5C domain has been extensively studied for a long time, but its catalytic mechanism, positional selectivity, and diverse substrate specificity remained mysterious due to the unavailability of three-dimensional structure. With the structural genomic approach, the first structure of IPP5C domain was solved from a S. pombe protein that is now known as SPsynaptojanin and the structure gave us answers to some of these questions.

Animals↗
PubMed1996

The genomic structure of the gene encoding the human transforming growth factor beta type II receptor (TGF-beta RII).

The genomic structure of the human transforming growth factor-beta type II receptor gene (TGF-beta RII) was determined by two PCR-based methods, the "long distance sequencer" method and the "promoter finder" method. Genomic fragments containing exons and adjacent introns were amplified by PCR, and the nucleotide sequences were determined by direct sequencing and subcloning sequencing. The TGF-beta RII protein is encoded by 567 codons in 7 exons. This is the first report about the genomic structure of a gene that belongs to the serine/threonine kinase type II receptor subfamily. Knowledge of the genomic structure of the TGF-beta RII gene will facilitate investigation of the TGF-beta signaling pathway in normal human cells and of the aberrations occurring during carcinogenesis.

Amino Acid Sequence↗
PubMed2004

Laboratory scale structural genomics.

At Lawrence Livermore National Laboratory, the development of the TB structural genomics consortium crystallization facility has paralleled several local proteomics research efforts that have grown out of gene expression microarray and comparative genomics studies. Collective experience gathered from TB consortium labs and other centers involved in the NIH-NIGMS protein structure initiative allows us to explore the possibilities and challenges of pursuing structural genomics on an academic laboratory scale. We discuss our procedures and protocols for genomic targeting approaches, primer design, cloning, small scale expression screening, scale-up and purification, through to automated crystallization screening and data collection. The procedures are carried out by a small group using a combination of traditional approaches, innovative molecular biochemistry approaches, software automation, and a modest investment in robotic equipment.

Cloning, Molecular↗
PubMed2000

Combining structural genomics and enzymology: completing the picture in metabolic pathways and enzyme active sites.

An important goal of structural genomics is to complete the structural analysis of all the enzymes in metabolic pathways and to understand the structural similarities and differences. A preliminary glimpse of this type of analysis was achieved before structural genomics efforts with the glycolytic pathway and efforts are underway for many other pathways, including that of catecholamine metabolism. Structural enzymology necessitates a complete structural characterization, even for highly homologous proteins (greater than 80% sequence homology), as every active site has distinct structural features and it is these active site differences that distinguish one enzyme from another. Short cuts with homology modeling cannot be taken with our current knowledge base. Each enzyme structure in a pathway needs to be determined, including structures containing bound substrates, cofactors, products and transition state analogs, in order to obtain a complete structural and functional understanding of pathway-related enzymes.

Binding Sites↗