IMPALA: matching a protein sequence against a collection of PSI-BLAST-constructed position-specific score matrices

MOTIVATION: Many studies have shown that database searches using position-specific score matrices (PSSMs) or profiles as queries are more effective at identifying distant protein relationships than are searches that use simple sequences as queries. One popular program for constructing a PSSM and comparing it with a database of sequences is Position-Specific Iterated BLAST (PSI-BLAST). RESULTS: This paper describes a new software package, IMPALA, designed for the complementary procedure of comparing a single query sequence with a database of PSI-BLAST-generated PSSMs. We illustrate the use of IMPALA to search a database of PSSMs for protein folds, and one for protein domains involved in signal transduction. IMPALA's sensitivity to distant biological relationships is very similar to that of PSI-BLAST. However, IMPALA employs a more refined analysis of statistical significance and, unlike PSI-BLAST, guarantees the output of the optimal local alignment by using the rigorous Smith-Waterman algorithm. Also, it is considerably faster when run with a large database of PSSMs than is BLAST or PSI-BLAST when run against the complete non-redundant protein database.     

A comparison of position-specific score matrices based on sequence and structure alignments

Sequence comparison methods based on position-specific score matrices (PSSMs) have proven a useful tool for recognition of the divergent members of a protein family and for annotation of functional sites. Here we investigate one of the factors that affects overall performance of PSSMs in a PSI-BLAST search, the algorithm used to construct the seed alignment upon which the PSSM is based. We compare PSSMs based on alignments constructed by global sequence similarity (ClustalW and ClustalW-pairwise), local sequence similarity (BLAST), and local structure similarity (VAST). To assess performance with respect to identification of conserved functional or structural sites, we examine the accuracy of the three-dimensional molecular models predicted by PSSM-sequence alignments. Using the known structures of those sequences as the standard of truth, we find that model accuracy varies with the algorithm used for seed alignment construction in the pattern local-structure (VAST) > local-sequence (BLAST) > global-sequence (ClustalW). Using structural similarity of query and database proteins as the standard of truth, we find that PSSM recognition sensitivity depends primarily on the diversity of the sequences included in the alignment, with an optimum around 30-50% average pairwise identity. We discuss these observations, and suggest a strategy for constructing seed alignments that optimize PSSM-sequence alignment accuracy and recognition sensitivity.     

Novel and unexpected bacterial diversity in an arsenic-rich ecosystem revealed by culture-dependent approaches

Background

BLAST is a commonly-used software package for comparing a query sequence to a database of known sequences; in this study, we focus on protein sequences. Position-specific-iterated BLAST (PSI-BLAST) iteratively searches a protein sequence database, using the matches in round i to construct a position-specific score matrix (PSSM) for searching the database in round i?+?1. Biegert and S?ding developed Context-sensitive BLAST (CS-BLAST), which combines information from searching the sequence database with information derived from a library of short protein profiles to achieve better homology detection than PSI-BLAST, which builds its PSSMs from scratch.

Results

We describe a new method, called domain enhanced lookup time accelerated BLAST (DELTA-BLAST), which searches a database of pre-constructed PSSMs before searching a protein-sequence database, to yield better homology detection. For its PSSMs, DELTA-BLAST employs a subset of NCBI??s Conserved Domain Database (CDD). On a test set derived from ASTRAL, with one round of searching, DELTA-BLAST achieves a ROC₅₀₀₀ of 0.270 vs. 0.116 for CS-BLAST. The performance advantage diminishes in iterated searches, but DELTA-BLAST continues to achieve better ROC scores than CS-BLAST.

Conclusions

DELTA-BLAST is a useful program for the detection of remote protein homologs. It is available under the ??Protein BLAST?? link at http://blast.ncbi.nlm.nih.gov.

Reviewers

This article was reviewed by Arcady Mushegian, Nick V. Grishin, and Frank Eisenhaber.     

SSALN: an alignment algorithm using structure-dependent substitution matrices and gap penalties learned from structurally aligned protein pairs

In template-based modeling of protein structures, the generation of the alignment between the target and the template is a critical step that significantly affects the accuracy of the final model. This paper proposes an alignment algorithm SSALN that learns substitution matrices and position-specific gap penalties from a database of structurally aligned protein pairs. In addition to the amino acid sequence information, secondary structure and solvent accessibility information of a position are used to derive substitution scores and position-specific gap penalties. In a test set of CASP5 targets, SSALN outperforms sequence alignment methods such as a Smith-Waterman algorithm with BLOSUM50 and PSI_BLAST. SSALN also generates better alignments than PSI_BLAST in the CASP6 test set. LOOPP server prediction based on an SSALN alignment is ranked the best for target T0280_1 in CASP6. SSALN is also compared with several threading methods and sequence alignment methods on the ProSup benchmark. SSALN has the highest alignment accuracy among the methods compared. On the Fischer's benchmark, SSALN performs better than CLUSTALW and GenTHREADER, and generates more alignments with accuracy >50%, >60% or >70% than FUGUE, but fewer alignments with accuracy >80% than FUGUE. All the supplemental materials can be found at http://www.cs.cornell.edu/ approximately jianq/research.htm.     

Alignments grow, secondary structure prediction improves.

Using information from sequence alignments significantly improves protein secondary structure prediction. Typically, more divergent profiles yield better predictions. Recently, various groups have shown that accuracy can be improved significantly by using PSI-BLAST profiles to develop new prediction methods. Here, we focused on the influences of various alignment strategies on two 8-year-old PHD methods. The following results stood out. (i) PHD using pairwise alignments predicts about 72% of all residues correctly in one of the three states: helix, strand, and other. Using larger databases and PSI-BLAST raised accuracy to 75%. (ii) More than 60% of the improvement originated from the growth of current sequence databases; about 20% resulted from detailed changes in the alignment procedure (substitution matrix, thresholds, and gap penalties). Another 20% of the improvement resulted from carefully using iterated PSI-BLAST searches. (iii) It is of interest that we failed to improve prediction accuracy further when attempting to refine the alignment by dynamic programming (MaxHom and ClustalW). (iv) Improvement through family growth appears to saturate at some point. However, most families have not reached this saturation. Hence, we anticipate that prediction accuracy will continue to rise with database growth.     

Strategies for the effective identification of remotely related sequences in multiple PSSM search approach

Searches using position specific scoring matrices (PSSMs) have been commonly used in remote homology detection procedures such as PSI-BLAST and RPS-BLAST. A PSSM is generated typically using one of the sequences of a family as the reference sequence. In the case of PSI-BLAST searches the reference sequence is same as the query. Recently we have shown that searches against the database of multiple family-profiles, with each one of the members of the family used as a reference sequence, are more effective than searches against the classical database of single family-profiles. Despite relatively a better overall performance when compared with common sequence-profile matching procedures, searches against the multiple family-profiles database result in a few false positives and false negatives. Here we show that profile length and divergence of sequences used in the construction of a PSSM have major influence on the performance of multiple profile based search approach. We also identify that a simple parameter defined by the number of PSSMs corresponding to a family that is hit, for a query, divided by the total number of PSSMs in the family can distinguish effectively the true positives from the false positives in the multiple profiles search approach.     

Use of multiple profiles corresponding to a sequence alignment enables effective detection of remote homologues

MOTIVATION: Position specific scoring matrices (PSSMs) corresponding to aligned sequences of homologous proteins are commonly used in homology detection. A PSSM is generated on the basis of one of the homologues as a reference sequence, which is the query in the case of PSI-BLAST searches. The reference sequence is chosen arbitrarily while generating PSSMs for reverse BLAST searches. In this work we demonstrate that the use of multiple PSSMs corresponding to a given alignment and variable reference sequences is more effective than using traditional single PSSMs and hidden Markov models. RESULTS: Searches for proteins with known 3-D structures have been made against three databases of protein family profiles corresponding to known structures: (1) One PSSM per family; (2) multiple PSSMs corresponding to an alignment and variable reference sequences for every family; and (3) hidden Markov models. A comparison of the performances of these three approaches suggests that the use of multiple PSSMs is most effective. CONTACT: ns@mbu.iisc.ernet.in.     

PSSMTS: position specific scoring matrices on tree structures

Identifying non-coding RNA regions on the genome using computational methods is currently receiving a lot of attention. In general, it is essentially more difficult than the problem of detecting protein-coding genes because non-coding RNA regions have only weak statistical signals. On the other hand, most functional RNA families have conserved sequences and secondary structures which are characteristic of their molecular function in a cell. These are known as sequence motifs and consensus structures, respectively. In this paper, we propose an improved method which extends a pairwise structural alignment method for RNA sequences to handle position specific scoring matrices and hence to incorporate motifs into structural alignment of RNA sequences. To model sequence motifs, we employ position specific scoring matrices (PSSMs). Experimental results show that PSSMs enable us to find individual RNA families efficiently, especially if we have biological knowledge such as sequence motifs. K. Sato and K. Morita contributed equally to this work.     

A novel method for protein secondary structure prediction using dual-layer SVM and profiles

A high-performance method was developed for protein secondary structure prediction based on the dual-layer support vector machine (SVM) and position-specific scoring matrices (PSSMs). SVM is a new machine learning technology that has been successfully applied in solving problems in the field of bioinformatics. The SVM's performance is usually better than that of traditional machine learning approaches. The performance was further improved by combining PSSM profiles with the SVM analysis. The PSSMs were generated from PSI-BLAST profiles, which contain important evolution information. The final prediction results were generated from the second SVM layer output. On the CB513 data set, the three-state overall per-residue accuracy, Q3, reached 75.2%, while segment overlap (SOV) accuracy increased to 80.0%. On the CB396 data set, the Q3 of our method reached 74.0% and the SOV reached 78.1%. A web server utilizing the method has been constructed and is available at http://www.bioinfo.tsinghua.edu.cn/pmsvm.     

PISCES: a protein sequence culling server

PISCES is a public server for culling sets of protein sequences from the Protein Data Bank (PDB) by sequence identity and structural quality criteria. PISCES can provide lists culled from the entire PDB or from lists of PDB entries or chains provided by the user. The sequence identities are obtained from PSI-BLAST alignments with position-specific substitution matrices derived from the non-redundant protein sequence database. PISCES therefore provides better lists than servers that use BLAST, which is unable to identify many relationships below 40% sequence identity and often overestimates sequence identity by aligning only well-conserved fragments. PDB sequences are updated weekly. PISCES can also cull non-PDB sequences provided by the user as a list of GenBank identifiers, a FASTA format file, or BLAST/PSI-BLAST output.     

Embedding strategies for effective use of information from multiple sequence alignments.

We describe a new strategy for utilizing multiple sequence alignment information to detect distant relationships in searches of sequence databases. A single sequence representing a protein family is enriched by replacing conserved regions with position-specific scoring matrices (PSSMs) or consensus residues derived from multiple alignments of family members. In comprehensive tests of these and other family representations, PSSM-embedded queries produced the best results overall when used with a special version of the Smith-Waterman searching algorithm. Moreover, embedding consensus residues instead of PSSMs improved performance with readily available single sequence query searching programs, such as BLAST and FASTA. Embedding PSSMs or consensus residues into a representative sequence improves searching performance by extracting multiple alignment information from motif regions while retaining single sequence information where alignment is uncertain.     

Sensitivity and selectivity in protein structure comparison

Seven protein structure comparison methods and two sequence comparison programs were evaluated on their ability to detect either protein homologs or domains with the same topology (fold) as defined by the CATH structure database. The structure alignment programs Dali, Structal, Combinatorial Extension (CE), VAST, and Matras were tested along with SGM and PRIDE, which calculate a structural distance between two domains without aligning them. We also tested two sequence alignment programs, SSEARCH and PSI-BLAST. Depending upon the level of selectivity and error model, structure alignment programs can detect roughly twice as many homologous domains in CATH as sequence alignment programs. Dali finds the most homologs, 321-533 of 1120 possible true positives (28.7%-45.7%), at an error rate of 0.1 errors per query (EPQ), whereas PSI-BLAST finds 365 true positives (32.6%), regardless of the error model. At an EPQ of 1.0, Dali finds 42%-70% of possible homologs, whereas Matras finds 49%-57%; PSI-BLAST finds 36.9%. However, Dali achieves >84% coverage before the first error for half of the families tested. Dali and PSI-BLAST find 9.2% and 5.2%, respectively, of the 7056 possible topology pairs at an EPQ of 0.1 and 19.5, and 5.9% at an EPQ of 1.0. Most statistical significance estimates reported by the structural alignment programs overestimate the significance of an alignment by orders of magnitude when compared with the actual distribution of errors. These results help quantify the statistical distinction between analogous and homologous structures, and provide a benchmark for structure comparison statistics.     

Prediction of protein relative solvent accessibility with a two-stage SVM approach

Information on relative solvent accessibility (RSA) of amino acid residues in proteins provides valuable clues to the prediction of protein structure and function. A two-stage approach with support vector machines (SVMs) is proposed, where an SVM predictor is introduced to the output of the single-stage SVM approach to take into account the contextual relationships among solvent accessibilities for the prediction. By using the position-specific scoring matrices (PSSMs) generated by PSI-BLAST, the two-stage SVM approach achieves accuracies up to 90.4% and 90.2% on the Manesh data set of 215 protein structures and the RS126 data set of 126 nonhomologous globular proteins, respectively, which are better than the highest published scores on both data sets to date. A Web server for protein RSA prediction using a two-stage SVM method has been developed and is available (http://birc.ntu.edu.sg/~pas0186457/rsa.html).     

PSP_MCSVM: brainstorming consensus prediction of protein secondary structures using two-stage multiclass support vector machines

Secondary structure prediction is a crucial task for understanding the variety of protein structures and performed biological functions. Prediction of secondary structures for new proteins using their amino acid sequences is of fundamental importance in bioinformatics. We propose a novel technique to predict protein secondary structures based on position-specific scoring matrices (PSSMs) and physico-chemical properties of amino acids. It is a two stage approach involving multiclass support vector machines (SVMs) as classifiers for three different structural conformations, viz., helix, sheet and coil. In the first stage, PSSMs obtained from PSI-BLAST and five specially selected physicochemical properties of amino acids are fed into SVMs as features for sequence-to-structure prediction. Confidence values for forming helix, sheet and coil that are obtained from the first stage SVM are then used in the second stage SVM for performing structure-to-structure prediction. The two-stage cascaded classifiers (PSP_MCSVM) are trained with proteins from RS126 dataset. The classifiers are finally tested on target proteins of critical assessment of protein structure prediction experiment-9 (CASP9). PSP_MCSVM with brainstorming consensus procedure performs better than the prediction servers like Predator, DSC, SIMPA96, for randomly selected proteins from CASP9 targets. The overall performance is found to be comparable with the current state-of-the art. PSP_MCSVM source code, train-test datasets and supplementary files are available freely in public domain at: and     

Benchmarking PSI-BLAST in genome annotation.

The recognition of remote protein homologies is a major aspect of the structural and functional annotation of newly determined genomes. Here we benchmark the coverage and error rate of genome annotation using the widely used homology-searching program PSI-BLAST (position-specific iterated basic local alignment search tool). This study evaluates the one-to-many success rate for recognition, as often there are several homologues in the database and only one needs to be identified for annotating the sequence. In contrast, previous benchmarks considered one-to-one recognition in which a single query was required to find a particular target. The benchmark constructs a model genome from the full sequences of the structural classification of protein (SCOP) database and searches against a target library of remote homologous domains (<20 % identity). The structural benchmark provides a reliable list of correct and false homology assignments. PSI-BLAST successfully annotated 40 % of the domains in the model genome that had at least one homologue in the target library. This coverage is more than three times that if one-to-one recognition is evaluated (11 % coverage of domains). Although a structural benchmark was used, the results equally apply to just sequence homology searches. Accordingly, structural and sequence assignments were made to the sequences of Mycoplasma genitalium and Mycobacterium tuberculosis (see http://www.bmm.icnet. uk). The extent of missed assignments and of new superfamilies can be estimated for these genomes for both structural and functional annotations.     

Scoring profile-to-profile sequence alignments

Sequence alignment profiles have been shown to be very powerful in creating accurate sequence alignments. Profiles are often used to search a sequence database with a local alignment algorithm. More accurate and longer alignments have been obtained with profile-to-profile comparison. There are several steps that must be performed in creating profile-profile alignments, and each involves choices in parameters and algorithms. These steps include (1) what sequences to include in a multiple alignment used to build each profile, (2) how to weight similar sequences in the multiple alignment and how to determine amino acid frequencies from the weighted alignment, (3) how to score a column from one profile aligned to a column of the other profile, (4) how to score gaps in the profile-profile alignment, and (5) how to include structural information. Large-scale benchmarks consisting of pairs of homologous proteins with structurally determined sequence alignments are necessary for evaluating the efficacy of each scoring scheme. With such a benchmark, we have investigated the properties of profile-profile alignments and found that (1) with optimized gap penalties, most column-column scoring functions behave similarly to one another in alignment accuracy; (2) some functions, however, have much higher search sensitivity and specificity; (3) position-specific weighting schemes in determining amino acid counts in columns of multiple sequence alignments are better than sequence-specific schemes; (4) removing positions in the profile with gaps in the query sequence results in better alignments; and (5) adding predicted and known secondary structure information improves alignments.     

A structure-based method for protein sequence alignment

MOTIVATION: With the continuing rapid growth of protein sequence data, protein sequence comparison methods have become the most widely used tools of bioinformatics. Among these methods are those that use position-specific scoring matrices (PSSMs) to describe protein families. PSSMs can capture information about conserved patterns within families, which can be used to increase the sensitivity of searches for related sequences. Certain types of structural information, however, are not generally captured by PSSM search methods. Here we introduce a program, Structure-based ALignment TOol (SALTO), that aligns protein query sequences to PSSMs using rules for placing and scoring gaps that are consistent with the conserved regions of domain alignments from NCBI's Conserved Domain Database. RESULTS: In most cases, the alignment scores obtained using the local alignment version follow an extreme value distribution. SALTO's performance in finding related sequences and producing accurate alignments is similar to or better than that of IMPALA; one advantage of SALTO is that it imposes an explicit gapping model on each protein family. AVAILABILITY: A stand-alone version of the program that can generate global or local alignments is available by ftp distribution (ftp://ftp.ncbi.nih.gov/pub/SALTO/), and has been incorporated to Cn3D structure/alignment viewer. CONTACT: bryant@ncbi.nlm.nih.gov.     

Structural/functional assignment of unknown bacteriophage T4 proteins by iterative database searches

Among the total of 274 orfs within bacteriophage T4, only half have been reasonably well characterized, and the functions of the rest have remained obscure. In order to predict the molecular functions of the orfs, a position-specific iterated (PSI)-BLAST search of bacteriophage T4 against the sequence database of known 3D structures was carried out. PSI-BLAST is one of the most powerful iterative sequence search methods using multiple sequence alignment, with the ability to detect many more proteins with distant homology than standard pairwise methods. The 3D structures of proteins are considered to be better preserved than the sequences, and the detected distantly homologous proteins are likely to possess highly similar 3D structures. Thirteen orfs of phage T4, whose homologues were not detected by standard pairwise methods, were found to have significantly homologous counterparts by this method. The plausibility of the results was confirmed by checking whether important residues at substrate/ligand-binding sites were conserved. Among them, two orfs, vs.1 and e.1, which are similar to Escherichia coli lytic enzyme and MutT protein, respectively, had not been studied previously. Also, gp rIIA, a rapid lysis protein, whose gene structure had been intensively studied during the development of molecular biology in the 1950s and yet whose molecular function remains unknown, has an N-terminal domain that is significantly similar to the N-terminal region of the heat shock protein Hsp90.     

Optimizing substitution matrices by separating score distributions

MOTIVATION: Homology search is one of the most fundamental tools in Bioinformatics. Typical alignment algorithms use substitution matrices and gap costs. Thus, the improvement of substitution matrices increases accuracy of homology searches. Generally, substitution matrices are derived from aligned sequences whose relationships are known, and gap costs are determined by trial and error. To discriminate relationships more clearly, we are encouraged to optimize the substitution matrices from statistical viewpoints using both positive and negative examples utilizing Bayesian decision theory. RESULTS: Using Cluster of Orthologous Group (COG) database, we optimized substitution matrices. The classification accuracy of the obtained matrix is better than that of conventional substitution matrices to COG database. It also achieves good performance in classifying with other databases.