Integration of structural and functional genomics

This paper introduces a special issue of Animal Genetics , which is devoted to the recent symposium held at Iowa State University entitled 'Integration of Structural and Functional Genomics'. We describe issues and needs that confront the animal genomics community, and describe how this symposium was structured to address these issues by improving communication and collaboration across species and disciplines. The session topics and oral presentations are briefly described for all invited speakers.     

The Protein Data Bank and structural genomics

The Protein Data Bank (PDB; http://www.pdb.org/) continues to be actively involved in various aspects of the informatics of structural genomics projects--developing and maintaining the Target Registration Database (TargetDB), organizing data dictionaries that will define the specification for the exchange and deposition of data with the structural genomics centers and creating software tools to capture data from standard structure determination applications.     

Protein family clustering for structural genomics

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.     

Structure-based functional inference in structural genomics

The dramatically increasing number of new protein sequences arising from genomics 4 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 [1–6], a considerable number of protein structures have already been produced, some of them coming directly out of semi-automated structure determination pipelines [6–10]. 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.     

Protein expression systems for structural genomics and proteomics

One of the key steps of structural genomics and proteomics is high-throughput expression of many target proteins. Gene cloning, especially by ligation-independent cloning techniques, and recombinant protein expression using microbial hosts such as Escherichia coli and the yeast Pichia pastoris are well optimized and further robotized. Cell-free protein synthesis systems have been developed for large-scale production of protein samples for NMR (stable-isotope labeling) and X-ray crystallography (selenomethionine substitution). Protein folding is still a major bottleneck in protein expression. Cell-based and cell-free methods for screening of suitable samples for structure determination have been developed for achieving a high success rate.     

Functional annotation by sequence-weighted structure alignments: statistical analysis and case studies from the Protein 3000 structural genomics project in Japan

A method to functionally annotate structural genomics targets, based on a novel structural alignment scoring function, is proposed. In the proposed score, position-specific scoring matrices are used to weight structurally aligned residue pairs to highlight evolutionarily conserved motifs. The functional form of the score is first optimized for discriminating domains belonging to the same Pfam family from domains belonging to different families but the same CATH or SCOP superfamily. In the optimization stage, we consider four standard weighting functions as well as our own, the "maximum substitution probability," and combinations of these functions. The optimized score achieves an area of 0.87 under the receiver-operating characteristic curve with respect to identifying Pfam families within a sequence-unique benchmark set of domain pairs. Confidence measures are then derived from the benchmark distribution of true-positive scores. The alignment method is next applied to the task of functionally annotating 230 query proteins released to the public as part of the Protein 3000 structural genomics project in Japan. Of these queries, 78 were found to align to templates with the same Pfam family as the query or had sequence identities > or = 30%. Another 49 queries were found to match more distantly related templates. Within this group, the template predicted by our method to be the closest functional relative was often not the most structurally similar. Several nontrivial cases are discussed in detail. Finally, 103 queries matched templates at the fold level, but not the family or superfamily level, and remain functionally uncharacterized.     

Protein crystallization in the structural genomics era

There are five broad areas where noteworthy advances have occurred in the field of macromolecular crystallization in the past 10 years, though some areas have seen the major part of those advances in only the last two years. This is largely a consequence of the international structural genomics initiative and its early results. The five areas are: (1) Physical studies and characterization of the protein crystallization process; (2) Development of new practical approaches and procedures; (3) The implementation of protein engineering by genetic means to enhance both purification and crystallization; (4) The creation of new screening conditions based on information and databases emerging from structural genomics; and (5) Development and implementation of automation, robotics, and mass screening of crystallization conditions using very small amounts of protein. A brief summary is provided here of the progress in the past few years and the influence of the structural genomics project.     

Finding functional sites in structural genomics proteins

Assigning function to structures is an important aspect of structural genomics projects, since they frequently provide structures for uncharacterized proteins. Similarities uncovered by structure alignment can suggest a similar function, even in the absence of sequence similarity. For proteins adopting novel folds or those with many functions, this strategy can fail, but functional clues can still come from comparison of local functional sites involving a few key residues. Here we assess the general applicability of functional site comparison through the study of 157 proteins solved by structural genomics initiatives. For 17, the method bolsters confidence in predictions made based on overall fold similarity. For another 12 with new folds, it suggests functions, including a putative phosphotyrosine binding site in the Archaeal protein Mth1187 and an active site for a ribose isomerase. The approach is applied weekly to all new structures, providing a resource for those interested in using structure to infer function.     

Metalloproteomics: high-throughput structural and functional annotation of proteins in structural genomics

A high-throughput method for measuring transition metal content based on quantitation of X-ray fluorescence signals was used to analyze 654 proteins selected as targets by the New York Structural GenomiX Research Consortium. Over 10% showed the presence of transition metal atoms in stoichiometric amounts; these totals as well as the abundance distribution are similar to those of the Protein Data Bank. Bioinformatics analysis of the identified metalloproteins in most cases supported the metalloprotein annotation; identification of the conserved metal binding motif was also shown to be useful in verifying structural models of the proteins. Metalloproteomics provides a rapid structural and functional annotation for these sequences and is shown to be approximately 95% accurate in predicting the presence or absence of stoichiometric metal content. The project's goal is to assay at least 1 member from each Pfam family; approximately 500 Pfam families have been characterized with respect to transition metal content so far.     

Gleaning structural and functional information from correlations in protein multiple sequence alignments

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Considerations in the identification of functional RNA structural elements in genomic alignments

Background  

Accurate identification of novel, functional noncoding (nc) RNA features in genome sequence has proven more difficult than for exons. Current algorithms identify and score potential RNA secondary structures on the basis of thermodynamic stability, conservation, and/or covariance in sequence alignments. Neither the algorithms nor the information gained from the individual inputs have been independently assessed. Furthermore, due to issues in modelling background signal, it has been difficult to gauge the precision of these algorithms on a genomic scale, in which even a seemingly small false-positive rate can result in a vast excess of false discoveries.     

FSSA: a novel method for identifying functional signatures from structural alignments

MOTIVATION: It is commonly believed that sequence determines structure, which in turn determines function. However, the presence of many proteins with the same structural fold but different functions suggests that global structure and function do not always correlate well. RESULTS: We propose a method for accurate functional annotation, based on identification of functional signatures from structural alignments (FSSA) using the Structural Classification of Proteins (SCOP) database. The FSSA method is superior at function discrimination and classification compared with several methods that directly inherit functional annotation information from homology inference, such as Smith-Waterman, PSI-BLAST, hidden Markov models and structure comparison methods, for a large number of structural fold families. Our results indicate that the contributions of amino acid residue types and positions to structure and function are largely separable for proteins in multi-functional fold families.     

Protein surface analysis for function annotation in high-throughput structural genomics pipeline

Structural genomics (SG) initiatives are expanding the universe of protein fold space by rapidly determining structures of proteins that were intentionally selected on the basis of low sequence similarity to proteins of known structure. Often these proteins have no associated biochemical or cellular functions. The SG success has resulted in an accelerated deposition of novel structures. In some cases the structural bioinformatics analysis applied to these novel structures has provided specific functional assignment. However, this approach has also uncovered limitations in the functional analysis of uncharacterized proteins using traditional sequence and backbone structure methodologies. A novel method, named pvSOAR (pocket and void Surface of Amino Acid Residues), of comparing the protein surfaces of geometrically defined pockets and voids was developed. pvSOAR was able to detect previously unrecognized and novel functional relationships between surface features of proteins. In this study, pvSOAR is applied to several structural genomics proteins. We examined the surfaces of YecM, BioH, and RpiB from Escherichia coli as well as the CBS domains from inosine-5'-monosphate dehydrogenase from Streptococcus pyogenes, conserved hypothetical protein Ta549 from Thermoplasm acidophilum, and CBS domain protein mt1622 from Methanobacterium thermoautotrophicum with the goal to infer information about their biochemical function.     

Integrative functional genomics

A report on the Keystone Symposium 'Biological Discovery Using Diverse High-Throughput Data', Steamboat Springs, USA, 30 March-4 April 2004.     

蚜虫基因组及功能基因组研究进展

蚜虫作为刺吸式昆虫和植物病毒的传播者,已经成为严重威胁农业生产发展的重要害虫之一。近几年随着分子生物学的发展,尤其是基因组测序技术的进步,蚜虫基因组学和功能基因组学取得了重大突破,使我们对蚜虫特殊的生物学特征有了深层次的认识。本文就蚜虫与内共生菌关系、表型可塑性、发育和生殖、系统进化、解毒酶基因家族以及唾液腺方面在基因组和功能基因组水平上的研究进展进行了综述。     

The CATH database: an extended protein family resource for structural and functional genomics

The CATH database of protein domain structures (http://www.biochem.ucl.ac.uk/bsm/cath_new) currently contains 34 287 domain structures classified into 1383 superfamilies and 3285 sequence families. Each structural family is expanded with domain sequence relatives recruited from GenBank using a variety of efficient sequence search protocols and reliable thresholds. This extended resource, known as the CATH-protein family database (CATH-PFDB) contains a total of 310 000 domain sequences classified into 26 812 sequence families. New sequence search protocols have been designed, based on these intermediate sequence libraries, to allow more regular updating of the classification. Further developments include the adaptation of a recently developed method for rapid structure comparison, based on secondary structure matching, for domain boundary assignment. The philosophy behind CATHEDRAL is the recognition of recurrent folds already classified in CATH. Benchmarking of CATHEDRAL, using manually validated domain assignments, demonstrated that 43% of domains boundaries could be completely automatically assigned. This is an improvement on a previous consensus approach for which only 10-20% of domains could be reliably processed in a completely automated fashion. Since domain boundary assignment is a significant bottleneck in the classification of new structures, CATHEDRAL will also help to increase the frequency of CATH updates.