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A canonical structural genomics programme is being conducted at the Paris-Sud campus area on baker's yeast proteins. Experimental strategies, first results and identified bottlenecks are presented. The actual or potential contributions to the structural genomics of several experimental structure-determination methods are discussed.
Structural genomics projects aim to solve the experimental structures of all possible protein folds. Such projects entail a conceptual shift from traditional structural biology in which structural information is obtained on known proteins to one in which the structure of a protein is determined first and the function assigned only later. Whereas the goal of converting protein structure into function can be accomplished by traditional sequence motif-based approaches, recent studies have shown that assignment of a protein's biochemical function can also be achieved by scanning its structure for a match to the geometry and chemical identity of a known active site. Importantly, this approach can use low-resolution structures provided by contemporary structure prediction methods. When applied to genomes, structural information (either experimental or predicted) is likely to play an important role in high-throughput function assignment.
Despite the advantages provided by the enormous recent increases in the availability of structural information, functional assignment for the large number of proteins represented in the sequence and structural genomics projects remains a pressing problem for genomic era biology. This section describes work relevant to this problem from several perspectives, including new approaches that take advantage of combined structure and sequence-based classification. Leveraging of genomic context and evolutionary information to improve classification and predictive power is a second prominent theme in the papers represented here. Finally, issues in building a database for linking sequence, structural, and functional information are explored.
A major goal of structural genomics is the provision of a structural template for a large fraction of protein domains. The magnitude of this task depends on the number and nature of protein sequence families. With a large number of bacterial genomes now fully sequenced, it is possible to obtain improved estimates of the number and diversity of families in that kingdom. We have used an automated clustering procedure to group all sequences in a set of genomes into protein families. Bench-marking shows the clustering method is sensitive at detecting remote family members, and has a low level of false positives. This comprehensive protein family set has been used to address the following questions. (1) What is the structure coverage for currently known families? (2) How will the number of known apparent families grow as more genomes are sequenced? (3) What is a practical strategy for maximizing structure coverage in future? Our study indicates that approximately 20% of known families with three or more members currently have a representative structure. The study indicates also that the number of apparent protein families will be considerably larger than previously thought: We estimate that, by the criteria of this work, there will be about 250,000 protein families when 1000 microbial genomes have been sequenced. However, the vast majority of these families will be small, and it will be possible to obtain structural templates for 70-80% of protein domains with an achievable number of representative structures, by systematically sampling the larger families.
The 2nd EMSL Workshop on Structural Genomics was held on 28th and 29th July 2000 at the Environmental Molecular Sciences Laboratory at the Department of Energy's Pacific Northwest National Laboratory in Richland, WA. The workshop focused on four topics: 1. The role for NMR in structural and functional genomics; 2. The technical challenges NMR faces for structural and functional genomics; 3. The potential need for a national NMR center for structural and functional genomics in the United States; and 4. Organization of the NMR community. This report summarizes the workshop proceedings and conclusions reached regarding the role of NMR in the emerging fields of structural and functional genomics.
MOTIVATION: Target selection strategies for structural genomic projects must be able to prioritize gene regions on the basis of significant sequence similarity with proteins that have already been structurally determined. With the rapid development of protein comparison software a robust prioritization scheme should be independent of the choice of algorithm and be able to incorporate different sequence similarity thresholds. RESULTS: A robust target selection strategy has been developed that can assign a priority level to all genes in any genome. Structural assignments to genome sequences are calculated at two thresholds and six levels (1-6) describe the prioritization of all whole genes and partial gene regions. This simple two-threshold approach can be implemented with any fold recognition or homology detection algorithms. The results for 10 genomes are presented using the SSEARCH and PSI-BLAST programs. AVAILABILITY: Programs are available on request from the authors.
We studied the genomic structure of human T-cell leukemia virus (HTLV) in the HTLV producer cell line MT-2. Southern blotting revealed that at least eight HTLV proviruses were integrated in the chromosomes of MT-2 cells. The genomic structure of these proviruses was analyzed using fragments of cloned HTLV that were specific to gag, pol, env, pXs and U3R genes as probes. We have identified a complete genome of HTLV in MT-2 (non-defective type). However, seven of the eight proviruses had defective genomes. Provirus T2-a contains only the U3R (LTR) of HTLV and T2-b corresponds to the non-defective genome. T2-c possesses only a portion of env, and pXs and U3R. T2-d consists of gag, pol, part of env and U3R. On the other hand, T2-e, f, g and h consist of gag, pXs and U3R. Northern blotting experiments with mRNA from MT-2 cells supported the evidence of amplification of the gag-pXs gene of HTLV. 26S mRNA is considered to be a subgenomic species of 35S RNA. 32S mRNA may represent the T2-d provirus which lacks a portion of env and pXs, while 20S mRNA was a subgenomic species. The gag-pXs gene may correspond to 24S mRNA, the amount which was amplified in MT-2 cells.
In structural genomics centers, nuclear magnetic resonance (NMR) screening is in increasing use as a tool to identify folded proteins that are promising targets for three-dimensional structure determination by X-ray crystallography or NMR spectroscopy. The use of 1D 1H NMR spectra or 2D [1H,15N]-correlation spectroscopy (COSY) typically requires milligram quantities of unlabeled or isotope-labeled protein, respectively. Here, we outline ways towards miniaturization of a structural genomics pipeline with NMR screening for folded globular proteins, using a high-density micro-fermentation device and a microcoil NMR probe. The proteins are micro-expressed in unlabeled or isotope-labeled media, purified, and then subjected to 1D 1H NMR and/or 2D [1H,15N]-COSY screening. To demonstrate that the miniaturization is functioning effectively, we processed nine mouse homologue protein targets and compared the results with those from the "macro-scale" Joint Center of Structural Genomics (JCSG) high-throughput pipeline. The results from the two pipelines were comparable, illustrating that the data were not compromised in the miniaturized approach.
Structural Genomics stands out among the emerging fields of proteomics since it influences the drug discovery process at so many points. Recent developments in protein expression technologies, x-ray crystallography and NMR spectroscopy provide the essential elements for high-throughput structure determination platforms. Bioinformatics methods to interrogate the resulting data will provide comprehensive, genome-wide databases of protein structure. Genomic sequencing and methods for high-throughput expression and protein purification are furthest advanced for microbial genes and so these have been the early targets for structural genomics initiatives. The information will be invaluable in understanding gene function, designing broad-spectrum small molecule inhibitors and in better understanding drug-host interactions.
Structural genomics efforts at the Chinese Academy of Sciences and Peking University are reported in this article. The major targets for the structural genomics project are targeted proteins expressed in human hematopoietic stem/progenitor cells, proteins related to blood diseases and other human proteins. Up to now 328 target genes have been constructed in expression vectors. Among them, more than 50% genes have been expressed in Escherichia coli, approximately 25% of the resulting proteins are soluble, and 35 proteins have been purified. Crystallization, data collection and structure determination are continuing. Experiences accumulated during this initial stage are useful for designing and applying high-throughput approaches in structural genomics.
Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-CeuI, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsed-field gel electrophoresis. We tested this experimental system on two representative bacterial genera: Salmonella and Pasteurella. Among Salmonella spp., I-CeuI mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the Salmonella spp. In marked contrast, the Pasteurella strains have very different genome structures among and even within individual species. The divergence of Pasteurella was also reflected in 16S rDNA sequences and far exceeded that seen between Escherichia and Salmonella. Based on this diversity, the Pasteurella haemolytica strains we analyzed could be divided into 14 phylogenetic groups and the Pasteurella multocida strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for Salmonella and Escherichia coli were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of P. multocida and P. haemolytica to be divided into different species, genera, or even higher ranks. On the other hand, strains of Pasteurella ureae and Pasteurella pneumotropica are very similar to those of P. multocida in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-CeuI-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.
Structural genomics is emerging as a principal approach to define protein structure-function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.
Currently, 119 high resolution structures of Thermotoga maritima proteins have been determined by the Joint Center for Structural Genomics (JCSG, www.jcsg.org). Sixty-seven of these were solved using the first implementation of the multi-tiered crystallization strategy at the JCSG for the efficient crystallization of large numbers of protein targets. Previously, we reported the analysis of all proteins crystallized using this multi-tiered strategy [Lesley, S.A. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 11664-11669; Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028-1037]. Here, we extend the analysis and describe the crystallization characteristics of those proteins that produced diffraction quality crystals, ultimately resulting in high resolution structures. First, we found that over 77% (52) of the crystals used for structure determination were produced directly from high-throughput coarse screens, indicating that less than one quarter of the crystals (15) required fine screening. In addition, as observed for the proteome screen [Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028-1037], the majority of conditions that produced crystals for natively expressed proteins, whose structures have been determined, were distinct from those of their more extensively purified and selenomethionine-labeled counterparts. Finally, 99% of the proteins whose structures were solved crystallized in conditions contained in the JCSG Minimal Core Screen [Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028-1037; Page, R. and Stevens, R.C. (2004) Methods 34, 373-389], a set of 67 conditions previously identified as those most likely to produce crystals of a diverse set of proteins, confirming its success for rapid identification of proteins with a natural propensity to crystallize.
The genome in a higher organism consists of a number of types of nucleotide sequence-specialized components, with each having tens of thousands of members or elements. It is crucial for our understanding of how a genome as an entity is organized, functions, and evolves to determine how these components are organized in the genome and how they relate with each other; however, no such knowledge is available. Here, we report a comprehensive analysis of the organization and interaction of all 40 components constituting the genome of the plant model species, Arabidopsis thaliana, at the whole-genome and chromosome levels. The 40 components include (i) 6 genome structural components consisting of GC%, genes, retrotransposons, DNA transposons, simple repeats, and low complex repeats; (ii) 3 evolutionarily critical features consisting of recombination rate, nucleotide substitutions, and nucleotide insertions/deletions; and (iii) 31 categories of genes with different functions and numbers of functions. We show that the distributions of 39 of the 40 components of the genome (excepting GC%) deviate significantly from the random distribution model and different types of the genome components are significantly correlated. These results remained to be true even when the genomic regions, such as centromeric regions, where transposable and repeat elements are abundant were excluded from the analyses. These findings suggest that DNA molecules contained in the Arabidopsis genome are each organized and structured from their constituting components in an unambiguous manner and that different types of the components that constitute or characterize the genome interact. The analysis also showed that each chromosome consists of a similar set of the components at similar densities, suggesting that the unique organization and interaction pattern of the components in each chromosome may represent, at least in part, the identity of a chromosome or a genome at the genome level, thus partly accounting for the phenotypic variation among different species. The data also provide comprehensive and new insights into many phenomena significant in genome biology, with which we particularly discuss the variation of genetic recombination. The variation of genetic recombination rate along a chromosomal arm is shaped, not only by the distribution of simple repeats, retrotransposons, DNA transposons, and nucleotide substitutions, but also by the functions of genes contained, especially those with multiple functions, suggesting that variation of genetic recombination along a chromosomal arm is the result of interactions among the components constituting local genome structure, function, and evolution.
Structural genomics projects represent major undertakings that will change our understanding of proteins. They generate unique datasets that, for the first time, present a standardized view of proteins in terms of their physical and chemical properties. By analyzing these datasets here, we are able to discover correlations between a protein's characteristics and its progress through each stage of the structural genomics pipeline, from cloning, expression, purification, and ultimately to structural determination. First, we use tree-based analyses (decision trees and random forest algorithms) to discover the most significant protein features that influence a protein's amenability to high-throughput experimentation. Based on this, we identify potential bottlenecks in various stages of the structural genomics process through specialized "pipeline schematics". We find that the properties of a protein that are most significant are: (i.) whether it is conserved across many organisms; (ii). the percentage composition of charged residues; (iii). the occurrence of hydrophobic patches; (iv). the number of binding partners it has; and (v). its length. Conversely, a number of other properties that might have been thought to be important, such as nuclear localization signals, are not significant. Thus, using our tree-based analyses, we are able to identify combinations of features that best differentiate the small group of proteins for which a structure has been determined from all the currently selected targets. This information may prove useful in optimizing high-throughput experimentation. Further information is available from http://mining.nesg.org/.
An important aspect of structural genomics is connecting coordinate data with whole-genome information related to phylogenetic occurrence, protein function, gene expression, and protein-protein interactions. Integrative database analysis allows one to survey the 'finite parts list' of protein folds from many perspectives, highlighting certain folds and structural features that stand out in particular ways.
As part of the Structural Proteomics In Europe (SPINE) project, the automated multifermenter system GRETA has been developed with structural genomics as a major application. GRETA comprises 6-24 parallel fermentation chambers, each with individual control of fermentation parameters such as temperature, stirring, pH, dissolved oxygen concentration and feed profiles. Six human proteins were used to optimize the GRETA fermentation processes and to compare these processes with typical baffled-flask protocols used in structural genomics projects. The optimized GRETA processes allows several times more protein to be produced per litre of culture with limited manual intervention and constitutes a potentially useful alternative both for scale-up production in structural proteomic projects and for fermentation-process optimization.
The genome size of Pseudoalteromonas haloplanktis, a ubiquitous and easily cultured marine bacterium, was measured as a step toward estimating the genome complexity of marine bacterioplankton. To determine total genome size, we digested P. haloplanktis DNA with the restriction endonucleases Notl and Sfil, separated the fragments using pulsed-field gel electrophoresis (PFGE), and summed the sizes of the fragments. The P. haloplanktis genome was 3512 +/- 112 kb by Notl digestion and 3468 +/- 54.1 kb by Sfil digestion. P. haloplanktis is also shown to have a complex genome structure, composed of two large replicons of approximately 2700 and 800 kb. Three pieces of evidence support this conclusion: (1) Two separate bands are always seen in PFGE of undigested P. haloplanktis DNA; (2) restriction digests of the larger band are missing a band of approximately 650 kb compared with restriction digests of total genomic DNA; and (3) a 16S rDNA probe hybridized to the larger replicon but not to the smaller. To our knowledge, P. haloplanktis is the first marine bacterium shown to have a complex genome structure.