Refining multiple sequence alignments with
conserved core regions
Saikat Chakrabarti, Christopher J. Lanczycki, Anna R. Panchenko, Teresa M. Przytycka,
Paul A. Thiessen and Stephen H. Bryant*
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda,
MD, 20894, USA
Received January 11, 2006; Revised February 19, 2006; Accepted April 3, 2006
Accurate multiple sequence alignments of proteins
are very important to several areas of computational
biology and provide an understanding of phylogen-
classification. This article presents a new algorithm,
REFINER, that refines a multiple sequence alignment
by iterative realignment of its individual sequences
with the predetermined conserved core (block)
model of a protein family. Realignment of each
sequence can correct misalignments between a
given sequence and the rest of the profile and at the
Large-scale benchmarking studies showed a notice-
able improvement of alignment after refinement. This
can be inferred from the increased alignment score
and enhanced sensitivity for database searching
using the sequence profiles derived from refined
alignments compared with the original alignments.
A standalone version of the program is available by
ftp distribution (ftp://ftp.ncbi.nih.gov/pub/REFINER)
and will be incorporated into the next release of the
Cn3D structure/alignment viewer.
The advent of large genome projects has led to an explosion of
sequence data in public databases. In this connection, the
establishment of structural, functional and evolutionary sim-
ilarity between proteins and protein domains is a challenging
task. Several domain databases are now available which com-
bine homologous protein domains into the distinct families
and represent them in the form of domain multiple sequence
alignments. The accuracy of domain identification, protein
classification and reconstruction of phylogenetic history of
domain families crucially depends on the quality of underlying
sequence alignments. Some domain resources, such as PFAM
(1) and ProDom (2), rely on the automated methods of mul-
CDD (4), employ careful manual intervention in constructing
the domain models. The CDD database contains alignments
that are carefully curated to be consistent with structure–
structure alignments to preserve the conserved core of a
protein domain family. Each curated CDD alignment records
conserved features within the family members in terms of
‘blocks’, the regions where every sequence is aligned without
Different methods have been proposed to produce a
multiple sequence alignment. Some of them align all
sequences simultaneously (5,6), while others apply a progress-
ive alignment strategy (7–10). According to the latter, the
sequences are aligned in a predetermined order dictated usu-
ally by the guide tree which groups similar sequences together
with the subsequent addition of more dissimilar ones. This
approach has been implemented in variety of programs and
packages such as MULTALIGN (11), MULTAL (9) and
CLUSTALW (10). While being widely accepted, progressive
alignment has its own pitfalls as the misalignment made at
previous stages can not be corrected afterwards and can
propagate into serious alignment errors. Moreover, the final
alignment strongly depends on the order of sequences being
aligned. To overcome these flaws, iterative approaches have
introduced the capacity to reconsider and realign previously
aligned sequences at each iteration with the goal of improving
in a random order and the iteration cycle ends as soon as a
convergence criterion has been satisfied. While this strategy
faces the problem of being trapped in a local minimum and
producing suboptimal alignment (like most other multiple
sequence alignment methods), it proves to be robust and
produces more accurate alignments (14,18–19).
In this article we present an algorithm named REFINER
that aims to refine an existing multiple alignment using the
*To whom correspondence should be addressed. Tel: 001 301 435 7792; Fax: 001 301 480 9241; E-mail: email@example.com
? The Author 2006. Published by Oxford University Press. All rights reserved.
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access
version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press
only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact firstname.lastname@example.org
Nucleic Acids Research, 2006, Vol. 34, No. 9
predetermined ‘block model’ of that domain family as a bio-
logically relevant constraint on the search space. A block
model represents conserved
which are highly unlikely to contain gaps and are common
to all family members. The refinement protocol works by
iterative random selection and realignment of sequences
with the familyblockmodeluntilthe alignment score saturates
to a stable value or until the iteration cycle terminates.
Realignment of each sequence can correct misalignments
between a given sequence and the rest of the profile by shifting
the individual blocks on that sequence and at the same time
preserves the family’s overall block model (i.e. the conserved
core regions). The latter constraint prohibits the insertion of
gap characters in the middle of conserved blocks. Following a
cycle of shifting for each sequence, the blocks in the block
model can also be extended or shortened in size depending on
the overall score improvement. The algorithm has been bench-
marked against structure-based, manually curated (CDD) and
un-curated (PFAM) alignments and shows an overall score
improvement. Comparison of the algorithm against another
independent alignment refinement method (19) showed better
performance. The refinement is further tested and validated by
checking the reliability in retrieval of functionally important
sites and enhanced sensitivity for profile-based database
searches compared with the original curated alignments.
This method is reasonably fast and can realign several hun-
dreds of highly diverse sequences within minutes. The refine-
ment method also provides a means to detect the outlier
sequences within an alignment and may thereby point a
way towards new subfamily identification schemes.
MATERIALS AND METHODS
A benchmark to evaluate the accuracy of
To test the overall performance of the refinement algorithm we
used a collection of 362 manually curated ‘parent alignments’
(set_362) from the CDD version 2.00 (4). The current version
of CDD is available at http://www.ncbi.nlm.nih.gov/Structure/
cdd/cdd.shtml. A parent alignment corresponds to the most
ancient (i.e. topmost) family in a domain family hierarchy,
several hundred of which are currently defined by CDD.
A smaller test set (set_94) of 94 multiple alignments from
CDD (with more than five protein structure entries) was
used to optimize the parameters for block extension cycles.
In addition, we applied the refinement algorithm also on
900 un-curated PFAM (1) alignments [generated either by
CLUSTALW (10) or T-Coffee (20)].
To compare the database search sensitivity of the Position
Specific Scoring Matrices (PSSMs) computed from multiple
sequence alignments before and after the refinement proced-
ure, we first constructed a list of true positives for the
conserved domain families from set_362. True positives
here are defined as those proteins/domains which are structur-
ally similar, as defined by the VAST algorithm (21,22), to the
representative structure of CDD alignments. First, for each
CDD alignment we chose a representative structure so that
the CDD footprint on this structure and corresponding
from MMDB structure database have been used (23)] were
consistent to a degree of 80% mutual overlap. By CDD foot-
print we mean the region on a structure between the first and
the last residues aligned in CDD. For CDD alignments that
have a corresponding MMDB structural domain, the VAST
structure neighbors of an MMDB domain/chain are retrieved
from the non-redundant set of MMDB chains. This set of 10
185 chains (db10185) was constructed by single-linkage clus-
tering, based on BLAST E-values of 10?80or less, from all the
entries in the MMDB structure database (23). Finally, we
checked the overlap of the CDD footprint and the VAST
footprint (the regions between first and last residues aligned
by VAST) for each structure from db10185. If the overlap
comprised at least 80% of each footprint length, then that
structure was recorded as being true positive and the VAST
footprint was recorded as a valid region. At the end of this
procedure 280 CDD family alignments (set_280) were selec-
ted which had at least one true positive entry.
We developed an algorithm that refines an existing alignment
by systematically realigning each sequence to better match the
profile constructed for the remaining sequences in the family.
The refinement is constrained by the block model implicit in
the family’s multiple alignment, where a block model com-
prises an ordered set of one or more non-overlapping blocks.
By definition, an aligned block in a multiple alignment is a
region containing no gaps on any sequence; it is specified
simply by a start position and a length in residues. An
unaligned region between blocks is called a loop. While we
discuss our algorithm in the context of a CDD multiple align-
ment that explicitly defines the conserved core region with a
block model, it applies more generally since it is straightfor-
ward to preprocess any multiple alignment, e.g. a PFAM
alignment, and derive a block model from its set of gap-free
columns. A tool to preprocess a FASTA file can be down-
loaded with the REFINER application itself.
The algorithm performs one or more iterations of refine-
ment, each of which in addition to iterative realignment of
individual sequences contains a phase of ‘block shifting’ fol-
lowed by a ‘block editing’ phase. The overall refinement
method is described in the flow chart (Figure 1). The engine
of the block shifting phase is a dynamic programming (DP)
module (24) that incorporates constraints to align a sequence
to the block model of a specified multiple alignment. Specific-
in the block model on a protein sequence, using the PSSM of
the specified multiple alignment to score each allowed
arrangement of the blocks on the sequence. The shifted blocks
retain the same relative order after DP. Since a block plays the
role of a single residue in traditional DP algorithms (e.g.
Smith–Waterman), the block-model-constrained DP we use
is very fast even for long alignments. To prevent spurious
long inserts, the distribution of loop sizes in the original
multiple alignment is used to further constrain the possible
block positions in the final alignment (24). In the results
described herein no loop in the refined alignment is allowed
to exceed the maximum loop length from the original
alignment. In the block shifting phase of the refinement, the
DP engine runs on each sequence of the original multiple
alignment to set new block positions. The order in which
Nucleic Acids Research, 2006, Vol. 34, No. 9 2599
the sequences are refined is randomized to avoid bias and
make the use of multiple iterations more effective. Conver-
gence is declared when no further improvement of overall
alignment score is observed or all iterations (maximum of
five iterations are used in our benchmarking studies) have
expired. The subsequent block editing phase examines each
block across all sequences in the multiple alignment to see if it
is advantageous to extend it at the N- and/or C-terminal end.
For this to be possible, the adjacent loop regions must contain
gap-free columns. Since this implies a block-length change,
we do not use the quantitative methods from the block shifting
phase to evaluate potential extensions. In the next section, we
introduce a 3-fold heuristic criteria used to control block
editing based on the statistical properties observed for block
and loop regions
Several scoring functions are used to evaluate the overall
alignment quality as well as the fitness of a particular sequence
to the existing profile.
For an alignment of length L the ‘row score’, Sr, is the sum
ofscores(derivedfromthePSSM)over allaligned positionsof
a particular row (sequence) r,
Here the PSSM is indexed by alignment column i and
the corresponding residues, AA from the sequence ‘r’. The
‘alignment score’, SN, for a multiple alignment with N rows
is simply the sum of all row scores:
SNis used as an objective function in the algorithm and Sris
employed by the DP engine to determine the refined block
positions for row r, and in that context care is taken to evaluate
Sragainst the PSSM computed from a multiple alignment
where row r is removed to avoid bias. Correcting an initial
misalignment of a particular sequence typically improves the
overall profile quality, measured in terms of SN.
During block editing, thescoresabove are notappropriate to
decide whether it is beneficial to add a new column(s) at a
block terminus because (i) the decision involves all sequences
of the alignment so that using Sris not relevant and (ii) adding
a new column would usually improve the overall score SN
whether truly beneficial or not. While large positive PSSM
values usually correspond to regions of conservation in a mul-
tiple alignment, small PSSM values do not necessarily imply
the location of badly aligned regions. Thus, we want to use a
measure to ensure addition of only informative columns yet
notoverlook columns containing well-conserved residues with
relatively small substitution scores.
To standardize the parameters for block extension we
examine all columns in blocks from CDD alignments in the
set_94 to establish heuristics based on observed differences
in PSSM values between alignment columns in blocks and
those that are not. Based on this analysis we developed
a 3-fold criterion used to evaluate block extension events
during refinement: after each block shifting phase, columns
in loop regions adjacent to blocks are scrutinized and added
to existing blocks until a column fails the 3-fold block
First, the distribution pattern of the median PSSM values for
residues in block-forming columns from set_94 indicates that
for a column to be added, the residues aligned in that column
should have a median PSSM matrix value of at least three (for
details see Results and Supplementary Data, Figure SM5).
Next, we examine both the frequency of occurrence of neg-
ative scores and the relative weight of negative PSSM values
in block-forming columns considering that the frequency and
relative weight of negative scores (unfavorable substitutions)
in the PSSM should be minimized for conserved block-
forming columns. Characterization of the block-forming
columns in set_94 suggests that the following threshold values
for these two additional parameters are characteristic of
alignment columns where block extension is predicted to be
beneficial. The frequency of negative scores for a block-
forming column, computed simply as the ratio of the number
of sequences with a negative PSSM value to the number of
alignment rows, should not exceed 0.3. The relative weight of
negative scores, computed as the absolute sum of negative
PSSM values in a column divided by the sum of all PSSM
values in that column, also should not exceed 0.3. It has been
observed that all the three criteria should be used together
to achieve better performance on block extension. Data
supporting these choices for block editing parameters are
presented in Supplementary Data (Figure SM5). The distribu-
tion of the PSSM values for non-block columns are found to be
Figure 1. Flowchart of the refinement algorithm.
2600Nucleic Acids Research, 2006, Vol. 34, No. 9
distinctively different from the block forming columns within
the dataset set_94 (data not shown).
Measuring the performance of refined alignment
If the block shifting phase of the refinement has an effect on
a sequence, the position of some of the blocks of the alignment
on that sequence must change. Changes in position of a
block ‘b’ have been recorded as a ‘relative block shift’ B,
where for a given sequence Dshiftis the difference in the posi-
tion of block N-terminus before and after the refinement and ‘b
is the length of the block.
The refined alignments are tested for their ability to
retrieve previously identified functionally important sites.
We used version 2.00 of the CDD alignment models to
collect information on the location of functionally important
sites that had been manually recorded by CDD curators
during detailed literature surveys. Nearly 7100 functionally
important sites (‘features’ in CDD alignments) were retrieved
from the set_362 multiple alignment models. The original
alignment of each of the functionally important columns
(FICs) was compared against that found after applying our
refinement method. This benchmarking study provides an
important standard of truth by which to gauge the accuracy
and quality of the alignments produced by our refinement
We used two different search methods, HMMER (25) and
SALTO_global (24) to test the ability of the refined sequence
profiles to find the corresponding VAST neighbors in the
dataset of 10 185 structural chains. The sensitivity–specificity
analysis was performed by calculating the receiver operating
characteristic (ROC) curves and ROC statistics. For a given
protein family one can calculate the fraction of detected true
positives and false positives at each similarity measure cut-off
(E-value for HMMER or raw score for SALTO_global). Sens-
itivity here is defined as a number of detected true positives
divided by the overall number of true positives in a database.
The specificity is calculated as a ratio between the number of
found false positives and the overall number of false positives
in the database. To compare sensitivities of profiles before and
after the refinement we measure the sensitivity at 1 and 5% of
false positive rates.
Improvement of alignment scores
The alignment refinement algorithm has been applied to 362
CDD multiple alignments and 900 PFAM (1) alignments
derived by both CLUSTALW (10) and T-Coffee (20) [See
Supplementary Data for lists]. Each sequence within these
alignments was realigned using the refinement procedure.
The overall alignment score has been calculated (Equation 2)
and the relative score improvement upon refinement is plotted
in Figure 2. As can be seen from this figure, in most cases for
both curated (75% of CDD alignments) and un-curated align-
ments (66 and 58% for CLUSTALW and T-Coffee derived
PFAM alignments) the alignment score has been improved
even without implementing the block editing phase of the
procedure, and the block editing phase improves the perform-
ance by additional 10–12%. However, higher occurrences of
negative improvement of alignment score are observed in
un-curated (CLUSTALW and T-Coffee) alignment compared
to curated (CDD) alignments. Our algorithm executes five
iterations (or less if converged) and in 70% of the cases the
overall score improves from iteration to iteration (Figure SM1
in Supplementary Data) which shows the effectiveness of
applying the iterative scheme in the refinement. It seems
that improvement of the alignment score largely depends on
the sequence diversity of the input alignments. Our results
indicate that 66% of all the improved alignments fall within
10–30% average sequence identity range (Figure SM2 in Sup-
It should be noted that the realignment of each sequence
generally improves both the fitness of that particular sequence
to the existing PSSM (row score, Equation 1) and the overall
alignment score (Equation 2). Grossly misaligned sequences
can therefore be detected by analyzing the difference in the
row score before and after the refinement. Although the detec-
ted misalignment can beautomatically corrected insomecases
(if the row score has been improved), there can be sequences
that do not belong to a given domain family (the row score can
go down). Hence, by applying the iterative refinement one can
identify such family outliers and remove them. As expected,
(a) and un-curated PFAM alignments (b) with or without block extension.
Nucleic Acids Research, 2006, Vol. 34, No. 92601
we showed that the number of such sequences is quite low
in manually curated CDD alignment (Supplementary Data,
Comparison of REFINER with other independent
We have compared the performance of our refinement method
(REFINER) with an iterative method for improvement of
multiple sequence alignment developed by Wallace et al.
(19). We applied their algorithm to our dataset (set_362)
and compared the improvement obtained by their and our
method. We applied the Remove first (RF) algorithm from
the Wallace et al. method using both average and log expecta-
tion (LE) scores to evaluate the alignments (19). Figure 3
shows the relative improvement of alignment (represented
by average score and LE score) obtained by the REFINER
over the method of Wallace et al. (19). For most cases (72 and
80% of CDs for average and LE scores, respectively) our
alignment refinement method provides better performance
in terms alignment score compared to that achieved by RF
Alignment score changes with respect to the block shift
To illustrate how the alignment score (Equation 2) changes
with respect to the magnitude of adjustments made in the
alignment, we plot the relative improvement of block score
(25% or higher) versus the block shift (Figure 4). The
block shift parameter is a convenient way to quantify the
changes in the alignment since the refinement algorithm shifts
individual sequences for a better alignment without altering
the length and number of blocks. As can be seen from this
figure the highest score improvement is only observed for a
small block shift of <10 residues. At the same time if the
alignment is considerably changed upon the refinement, the
overall score almost does not increase. This may indicate that
moderate adjustments of the curated alignments made by the
refinement algorithm are almost always beneficial while large
alignment shifts may be detrimental given how reasonable the
original structure-based (CDD) alignments are. This is further
supported by the observation of similar correlation between
the improvement of overall alignment score and the block shift
(Figure SM4, Supplementary Data). The examination of
scores of individual blocks before and after the refinement
can also be useful in identifying the problematic blocks
which either should be removed or realigned manually.
The extension of the blocks, as previously mentioned, may
also improve the overall alignment score; altogether 25%
of all blocks from 68% of CDD alignments can undergo
extension. Figure 5 shows that adding one or two residues
at the ends of blocks does not lead to a score increase whereas
more extensive editing of block boundaries (up to 10 residues
at either block terminus) can improve the alignment score.
Stretching the blocks over the whole alignment would yield
very small improvement as well. This in turn implies that the
current CDD block model is specific enough for a given family
but there are conserved columns in the inter-block regions
which should also be taken into account.
Figure 3. Comparison of performance of refinement. Histograms of relative improvement of alignments score achieved by our refinement method
(REFINER) over RF algorithm of Wallace et al. (19) refinement package. Alignments are evaluated by both average and LE scores (19). Relative improvement
of alignment score is measured as the difference between the final scores after application of REFINER and RF method divided by the final score obtained by RF
2602Nucleic Acids Research, 2006, Vol. 34, No. 9
The accuracy of the refinement algorithm has been tested
further in terms of the retrieval of previously identified
FICs from set_362 structure-based, curated alignments. We
assume that the FICs should not change much upon the refine-
ment since they represent manually annotated conserved sites
for a family and can be used as a standard of truth. It has been
shown previously that fully automated procedure of functional
site prediction can recover most of the annotated FIC from
CDD for similar but not identical test set (26). FICs were
extracted (see Materials and Methods) from the original
CDD alignments and compared against that derived after
applying the refinement on the same CDD alignment.
Figure 6 shows that majority (?70%) of FICs remain
unchanged after the refinement; i.e. the refinement algorithm
does not perturb and successfully reproduces the exact align-
ment for important residues. We also carefully examined those
cases where the refinement algorithm introduced some
changes in the alignment of FICs. In fact, 67% of changed
FICs have ahigherscore after therefinementascomparedwith
the original alignment (Figure 6 inset), indicating some
improvement is possible even at these sites.
Figure 7 shows an example of the alignment of the members
of Bowman–Birk type proteinase inhibitor (BBI) family
before andafter the refinement.FICsare highlighted byyellow
in the figure. While the refinement algorithm improves the
overall score for the whole alignment (?5%), it also changes
the alignment of FICs so that score of some FICs increases by
24%. As can be seen from this figure the score increase is
caused by the correction of misaligned Cys residues which
form the disulfide bond in all the representative structures of
this family. Therefore, in this case the refinement algorithm
was able to not just reproduce the FICs but also improve the
alignment for some of them.
Comparison of the sensitivity/specificity of PSSMs
before and after the refinements
One way to validate the multiple alignment method is to
examine its performance of produced alignment or sequence
profile in homology-based database searches. We used two
Figure 4. Effect of block shift on block score. The relative improvement (using structure-based, curated CDD alignments) of score of 25% or higher per block is
plotted versus the block shift. The central line in each box shows the median value, the upper and lower boundaries of individual box show the upper and lower
the percentage of data points for each block shift bin.
Figure 5. Effect of block extension on alignment score. The relative improve-
ment of alignment score (AS, Equation 2) is shown for each bin of the block
all blocks within a CDD alignment.
Nucleic Acids Research, 2006, Vol. 34, No. 92603
methods to perform the database searches namely HMMER
and SALTO_global (24). The HMM models were constructed
using global mode of HMMER 2.3.2 (25). SALTO_global
represents a global version of SALTO algorithm presented
in the Materials and Methods section (24) and requires all
blocks to be aligned in the final multiple sequence alignment.
Each HMM model and PSSM derived from the alignments
before and after the refinement procedure were used to search
the ‘non-redundant’ database of protein chains (db10185).
The sensitivities of finding VAST neighbors by two search
methods HMMER and SALTO_global at 1 and 5% error
rates are given in Table 1. It is clear from the table that the
sensitivities of sequence profiles/HMMs have increased upon
the refinement; this result is more pronounced when the
HMMER is used to search the database. Although the overall
improvement in sensitivity is not dramatic (?5%) it implies
that the refinement algorithm produces better alignments. To
investigate this further we calculated the correlation between
the relative improvement in alignment score and relative
improvement in sensitivity (Table 2). This table shows that
there is statistically significant correlation between the
Figure 6. Quality control by testing the recovery of FIC. Alignments of FICs
are compared before and after the refinement. The automated refinement pro-
cedure could reproduce the exact same alignment that was obtained by careful
manual curation for most (shown by black box) of the FICs. In addition,
majority of the changed FICs show better score (inset, improvement+) when
compared against the score derived before the refinement.
Figure 7. Improvement of alignment after refinement. Alignments of Bowman-Birk type proteinase inhibitor (BBI) family (CDD code: cd00023) derived
(a) before and (b) after the refinement show marked improvement. Block forming columns are displayed in capitals where functional important residues are
boxedinyellow.Oneoftheconservedcysteinesitesisshownin redin (b)and(c)whereprobablemisalignmentsarecorrectedin numberofsequences.(c) Displays
backbone representation of the structure of hydrolase inhibitor (pdb code: 1C2A). Functional important sites are marked in yellow and disulfide bonds are shown
2604 Nucleic Acids Research, 2006, Vol. 34, No. 9
alignment score and sensitivity, where the latter is being used
as an indication of alignment accuracy. Even though database
search methods may not be sensitive enough to capture the
entire difference between the profiles before and after the
refinement, we can conclude that the improvement observed
in the alignment score can be the result of the alignment
improvements made by the refinement algorithm.
We developed a new algorithm to refine a multiple alignment
ofproteinsequences.Our approach assumes thatthealignment
is represented by a set of conserved regions or blocks that are
aligned without gaps. One example of such a block-based
alignment constitutes the structure-based, manually curated
CDD alignment, which can be used to infer the domain organ-
ization, predict functional sites or model structures of
unknown proteins. In this case the refinement algorithm can
be used as a tool to assist CDD curation and as a standalone
programtorefineamultiplesequence alignment.Wehave also
applied our refinement algorithm on un-curated multiple
alignments (PFAM dataset) derived by CLUSTALW (10)
and T-Coffee (20).
In this article we demonstrated that our refinement
algorithm improves the input alignment not only when it is
applied to un-curated alignment but also to a curated align-
ment. Since the input alignment represents a rather accurate
alignment and assumes block structure, we have to develop
sensitive, dedicated methods to measure the alignment
improvement. Therefore, in addition to measuring the
improvement by alignment score increase we analyzed the
statistics of block extensions and shifts, quality of recovering
FICs and sensitivity of sequence profiles based on refined
alignments. We showed that the refinement algorithm
shows an improvement with respect to all these measures.
We also showed better performance of our method
when compared with an iterative alignment refinement
In summary, our approach provides a fast and accurate
method for refinement of existing block-based alignments.
In particular, this method can be used to refine alignments
by correcting local misalignments and to find sequence out-
liers, which do not fit the domain family model.
Supplementary data are available at NAR Online.
The authors thank Dr Maricel G. Kann for kindly providing a
standalone version of SALTO_global program. This work was
supported by the Intramural Research Program of the National
Library of Medicine at National Institutes of Health/DHHS.
Funding to pay the Open Access publication charges for this
article was provided by Intramural Research Program of the
National Library of Medicine at National Institutes of Health/
Conflict of interest statement. None declared.
1. Bateman,A., Birney,E., Cerruti,L., Durbin,R., Etwiller,L., Eddy,S.R.,
Griffiths-Jones,S., Howe,K.L., Marshall,M. et al. (2002) The Pfam
protein families database. Nucleic Acids Res., 30, 276–280.
2. Servant,F., Bru,C., Carrere,S., Courcelle,E., Gouzy,J., Peyruc,D. and
Kahn,D (2002) ProDom: Automated clustering of homologous domains.
Brief. Bioinformatics, 3, 246–251.
3. Letunic,I., Goodstadt,L., Dickens,N.J., Doerks,T., Schultz,J., Mott,R.,
Ciccarelli,F., Copley,R.R., Ponting,C.P. and Bork,P. (2002) Recent
improvements to the SMART domain-based sequence annotation
resource. Nucleic Acids Res., 30, 242–244.
4. Marchler-Bauer,A., Panchenko,A.R., Shoemaker,B.A., Thiessen,P.A.,
Geer,L.Y. and Bryant,S.H. (2002) CDD: a database of conserved
domain alignments with links to domain three-dimensional structure.
Nucleic Acids Res., 30, 281–283.
5. Lipman,D.J., Altschul,S.F. and Kececioglu,J.D. (1989) A tool for
multiple sequence alignment. Proc. Natl Acad. Sci. USA, 86,
6. Stoye,J., Moulton,V. and Dress,A.W. (1997) DCA: an efficient
implementation of the divide-and-conquer approach to simultaneous
multiple sequence alignment. Comput Appl Biosci., 13, 625–626.
7. Hogeweg,P. and Hesper,B. (1984) The alignment of sets of sequences
and the construction of phyletic trees: an integrated method. J. Mol.
Evol., 20, 175–186.
8. Feng,D.F. and Doolittle,R.F. (1987) Progressive sequence alignment as
a prerequisite to correct phylogenetic trees. J. Mol. Evol., 25, 351–360.
9. Taylor,W.R (1988) A flexible method to align large numbers of
biologicalsequences. J. Mol. Evol., 28, 161–169.
10. Thompson,J.D., Higgins,D.G. and Gibson,T.J. (1994) CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and weight
matrix choice. Nucleic Acids Res., 22, 4673–4680.
11. Barton,G.J. and Sternberg,J.E. (1987) A strategy for the rapid multiple
alignment of protein sequences. Confidence levels from tertiary
structure comparisons. J. Mol. Biol., 198, 327–337.
12. Gotoh,O. (1996) Significant improvement in accuracy of multiple
protein sequence alignments by iterative refinement as assessed by
reference to structural alignments. J. Mol. Biol., 264, 823–838.
13. Notredame,C. and Higgins,D.G. (1996) SAGA: sequence alignment by
genetic algorithm. Nucl. Acids Res., 24, 1515–1524.
14. Heringa,J. (2002) Local weighting schemes for protein multiple
sequence alignment. Comput. Chem., 26, 459–477.
Table 1. Sensitivity values estimated from the ROC curves at 1 and 5% error
rates (fraction of false positives)
Search method Error rate Before_Refiner After_Refiner After_Refiner_exta
aRefiner with BLOCK extension.
Table 2. Correction between the relative improvement of alignment score and
Search methodPSSM usedCorrelation cofficient
At 1% FP At 5% FP
All values are shown over the increases from search derived with original
(before refinement) alignments.
*Statistical significance or P-values < 0.05.
Nucleic Acids Research, 2006, Vol. 34, No. 92605
15. Berger,M.P. and Munson,P.J. (1991) A novel randomized iterative Download full-text
strategy for aligning multiple protein sequences. Comput. Appl. Biosci.,
16. Katoh,K., Misawa,K., Kuma,K. and Miyata,T. (2002) MAFFT: a novel
method for rapid multiple sequence alignment based on fast Fourier
transform. Nucleic Acids Res., 30, 3059–3066.
17. Do,C.B., Mahabhashyam,M.S., Brudno,M. and Batzoglou,S. (2005)
ProbCons: probabilisticconsistency-based multiplesequence alignment.
Genome Res., 2, 330–340.
18. Wang,Y. and Li,K.B. (2004) An adaptive and iterative algorithm for
refining multiple sequence alignment. Comput. Biol. Chem., 28,
19. Wallace,I.M., O’Sullivan,O. and Higgins,D.G (2005) Evaluation of
iterative alignment algorithms for multiple alignment. Bioinformatics,
20. Notredame,C., Higgins,D.G. and Heringa,J. (2000) T-Coffee: a novel
method for fast and accurate multiple sequence alignment. J. Mol. Biol.,
21. Madej,T., Gibrat,J-F. and Bryant,S.H. (1995) Threading a database of
protein cores. Protein Struct. Funct. Genet., 23, 356–369.
22. Gibrat,J.F., Madej,T. and Bryant,S.H. (1996) Surprising similarities
in structure comparison. Curr. Opin. Struct. Biol., 6,
23. Chen,J., Anderson,J.B., DeWeese-Scott,C., Fedorova,N.D., Geer,L.Y.,
He,S., Hurwitz,D.I., Jackson,J.D., Jacobs,A.R., Lanczycki,C.J. et al.
(2003) MMDB: Entrez’s 3D-structure database. Nucleic Acids Res., 31,
24. Kann,M.G., Thiessen,P.A., Panchenko,A.R., Schaffer,A.A.,
Altschul,S.F. and Bryant,S.H. (2005) A structure-based method
for protein sequence alignment. Bioinformatics, 21,
25. Eddy,S.R. (1998) Profile hidden Markov models. Bioinformatics, 14,
26. Panchenko,A.R., Kondrashov,F. and Bryant,S. (2004) Prediction of
functional sites by analysis of sequence and structure conservation.
Protein Sci., 13, 884–892.
2606 Nucleic Acids Research, 2006, Vol. 34, No. 9