Expresso: automatic incorporation of structural
information in multiple sequence alignments
using 3D-Coffee
Fabrice Armougom, Se´bastien Moretti, Olivier Poirot, Ste´phane Audic, Pierre Dumas1
,
Basile Schaeli1
, Vladimir Keduas and Cedric Notredame*
Laboratoire Information Ge´nomique et Structurale, CNRS UPR2589, Institute for Structural Biology and Microbiology
(IBSM), Parc Scientifique de Luminy, 163 Avenue de Luminy, FR- 13288, Marseille cedex 09, France and 1
Laboratoire
de syste`mes pe´riphe´riques, Ecole Polytechnique Fe´de´rale de Lausanne, CH 1015 Lausanne, Switzerland
Received February 14, 2006; Revised March 1, 2006; Accepted March 7, 2006
ABSTRACT
Expresso is a multiple sequence alignment server
that aligns sequences using structural information.
The user only needs to provide sequences. The server
runs BLAST to identify close homologues of the
sequences within the PDB database. These PDB
structures are used as templates to guide the alignment
of the original sequences using structure-based
sequence alignment methods like SAP or Fugue.
The final result is a multiple sequence alignment of
the original sequences based on the structural
information of the templates. An advanced mode
makes it possible to either upload private structures
or specify which PDB templates should be used
to model each sequence. Providing the suitable structural
information is available, Expresso delivers
sequence alignments with accuracy comparable
with structure-based alignments. The server is available
on http://www.tcoffee.org/.
INTRODUCTION
Over the past years, multiple sequence alignments (MSAs)
have become one of the most widely used tools in biology
along with database search methods. MSAs are needed
for profile analysis, phylogenetic reconstruction, structure
prediction and a wealth of minor but important applications
such as PCR primer design or sequence reconciliation. The
ever-growing reliance on MSAs is even more pronounced now
that hundreds of complete genomes are being made available.
This window opened on evolution provides an ideal context
for MSAs to fulfill their potential as key tools in functional
genomics.
Unfortunately, MSA packages are not yet accurate enough
to deliver all their promises and the sharp increase in the
number of methods recently published (25 novel programs
over the last 5 years) illustrates well the expectation for
improvement within the community. MSAs are not always
good enough for large-scale analysis and while immense
progress has been made to accurately align multiple sets of
sequences with >40% average identity, recent benchmarks
published with the MAFFT 5 package (1) reveal that state
of the art methods still fail to reliably align distantly related
sequences. In the so-called ‘Twilight zone’ (2), sequences with
<20% identity cannot be aligned with >30% average accuracy
(as judged by comparison with reference alignments). So far,
the most convincing solution to this problem has been to
supplement sequences with structural information (3).
The reason why structure-based MSAs are more accurate is
not so much a consequence of better algorithms but rather an
effect of structures evolutionary stability. Structures evolve
slower than the sequences (4) and even when sequences
have diverged beyond recognition it is often possible to establish
homology (i.e. common ancestry) on the basis of 3D folds
comparisons (3). The increasing availability of structural data
(5) means that relying on structure-based methods for
sequence analysis has become much more realistic than it
used to be. However, sequences are still being determined
much faster than structures, thus creating a context where
methods able to efficiently combine sequences and structure
into accurate MSAs are needed. To the best of our knowledge,
only six algorithms have been designed that are able to make
use of secondary (6,7) or tertiary (8–10) structure information.
In the context of this work, we used 3D-Coffee (11) for its
ability to combine the output of several methods into one
unique model. 3D-Coffee is based on the T-Coffee algorithm
(12), a heuristic method that uses a progressive algorithm to
compute an MSA having a high consistency with a collection
of pre-computed pairwise alignments (the library).
*To whom correspondence should be addressed. Tel: +33 491 825 427; Fax: +33 491 825 420; Email: cedric.notredame@europe.com
Ó The Author 2006. Published by Oxford University Press. All rights reserved.
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doi:10.1093/nar/gkl092
In 3D-Coffee, the principle is the same except that the library’s
pairwise alignments are derived from structural superpositions
using methods like Sap (13), Lsqman (14) or possibly any
alternative structure alignment package [for a review see
(15)]. When using combinations of structures and sequences,
3DCoffee can also incorporate structure-sequence (threading)
alignment methods like Fugue (16) to ease the diffusion of
structural information onto the sequences.
3D-Coffee has been available via the web server
3DCoffee@igs for >2 years (17). The original implementation
made it possible to combine sequences and structures using
the most advanced T-Coffee options through a simple web
interface. Although it provides access to most of the T-Coffee
inline functions, this server requires the user to explicitly
specify which structural template is to be associated with
each sequence. This specification, made through a cumbersome
procedure of sequence renaming, was complicated and
impractical for non-specialists.
The novel version of 3D-Coffee@igs is named Expresso
because it makes it possible for non-specialists to rapidly and
automatically benefit from the strength of 3D-Coffee. The
term Expresso also conveys the notion of aroma extraction
and concentration, a notion that resonates with the way structures
are ‘expressed’ within the MSA. In Expresso, we implemented
an automated identification of suitable structural
templates via a BLAST search against the PDB database.
3D-Coffee uses the selected structures to assemble a genuine
structure-based MSA during a process that merely looks like a
standard sequence alignment procedure from the user’s point
of view. Providing the appropriate structural information is
available, Expresso is significantly more accurate than regular
homology-based methods and its alignments are often indistinguishable
from reference structure-based alignments (11).
METHODS
Selection of the Structural Template
The core idea of Expresso is to reliably identify structures that
can be used as templates for the sequences (source) one wishes
to align. The rationale is that any alignment carried out on the
templates can easily be transposed onto the source sequences
as long as the source and the template are highly homologous.
The most basic and important step in Expresso is a BLAST
search of the source sequences against PDB, in order to
identify suitable templates. A BLAST match is considered
a suitable template if it displays a minimum of 60% sequence
identity with the source sequence and a minimum coverage of
70% (i.e. 70% of the source sequence residues matched).
These rather conservative criteria were chosen to limit the
template selection to close homologues whose alignment
with the source is entirely non-ambiguous. No effort is
made to identify structures with special conformations, or
resolutions, although this could easily be added to the pipeline.
However, whenever the automatic procedure appears inappropriate,
the user can explicitly declare the source–template
association using the advanced mode of the server.
Integration of the Structural Template
Once every sequence with a structural homologue has
been assigned its template, 3DCoffee undertakes the
library computation step. It applies a collection of
pre-defined pairwise alignment methods on every pair of
sequences. The methods are either sequence-based alignment
(e.g. lalign) or structure-based (e.g. SAP). When using
structural methods, a structure-based alignment of the templates
is first computed. The two source sequences are
then aligned to their respective templates, and the induced
pairwise alignment of the two sources is integrated within
the library (Figure 1). The accuracy of this delicate process
relies on a high level of identity between the source and the
template sequence, hence the stringency of the original
BLAST search.
Alignment computation
Once the library assembly step is finished, the MSA is
assembled in a progressive fashion, using the standard
T-Coffee algorithm. The default mode of the server for
running T-Coffee is
t_coffee <seq> -in Mslow_pair, Msap_pair, Mlalign_
id_pair –template_file SCRIPT_blast.pl,
where SCRIPT_blast.pl is a stand-alone script that BLASTs
every source sequence against PDB in order to identify suitable
structural templates (if they exist).
USING EXPRESSO
Default mode
The server can be accessed at http://www.tcoffee.org/, by
clicking on the Expresso link, either advanced or regular.
To use the regular mode, one simply needs to cut and paste
FASTA sequences. No special precaution is needed to name
the sequences.
Advanced mode
The advanced mode of the server offers many more possibilities
and guides the user with a series of bulleted points:
 Cut and paste your sequences.
 Upload your PDB structures. Should be used when
some of the structures are not in the public domain. When
uploading a PDB template, the associated source sequence
is automatically generated using the SEQRES field. PDB
files must follow the standard PDB format and the server
requires a TITLE, a HEADER, an ATOM and a SEQRES
section.
 Select the methods. The default selection corresponds to
3DCoffee. Further structure alignment methods will soon
be added, along with new multiple sequence alignment
packages. Users are welcome to suggest the incorporation
of any public domain method.
 PDB template selection. By default no template is used in
the advanced mode. Users should check the SCRIPT box to
automatically fetch the templates with BLAST, or specify
the source to template correspondences in the box below.
The format for doing so is indicated in the corresponding
section.
Figure 2 shows a typical output, computed on the
HOMSTRAD thioredoxin family (18). The first alignment
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(Figure 2a) was computed using the standard T-Coffee
protocol, while the other (Figure 2b) is an Expresso
MSA computed using the regular mode. In the T-Coffee
alignment, 15% of the columns are correctly aligned (as
judged by comparison with the HOMSTRAD reference
alignment) while in the Expresso MSA, 49% of the columns
appear to be correct. Figure 2c shows which template was
selected for each sequence. When selecting the template,
no attempt is made to match the source sequence name
with the template name, which sometimes results
in apparent discrepancies (1aaza modelled with 1de2A).
While in most cases, these arbitrary choices should
not affect the output, better control can be achieved by
specifying the template/sequence correspondence in the
advanced mode.
CONCLUSION AND FUTURE DEVELOPMENTS
Expresso is an improved version of the original 3DCoffee@igs
server. Structures are now fetched automatically and used to
guide the alignment. This procedure can result in a dramatic
improvement of the sequence alignment when homologue
PDB structures are available. From the user point of view,
Expresso is a regular multiple sequence alignments server that
seamlessly includes structural information in MSAs, allowing
Figure 1. Computation of a template-based library. Structural templates are assigned to each original source sequence and these templates are used to generate a
structure-based sequence alignment. The final library alignment is generated by aligning each source sequence with its template, thus generating a template-based
alignment of the two sources.
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non-specialists to benefit from the power of structure-based
sequence alignment without having to address all the technical
issues it implies. Future developments will involve a gradual
extension of the methods available for combination in the
advanced section.
We strongly encourage users to send us their feedback.
ACKNOWLEDGEMENTS
We thank Prof. Jean-Michel Claverie (head of IGS) for stimulating
scientific discussions and material support. We also
thank Prof. Roger Hersch (EPFL) for useful advices on code
optimization. The development of the server was supported by
CNRS (Centre National de la Recherche Scientifique),
Sanofi-Aventis Pharma SA., Marseille–Nice Ge´nopole and
the French National Genomic Network (RNG). Funding to
pay the Open Access publication charges for this article was
provided by CNRS.
Conflict of interest statement. None declared.
REFERENCES
1. Katoh,K., Kuma,K., Toh,H. and Miyata,T. (2005) MAFFT version 5:
improvement in accuracy of multiple sequence alignment. Nucleic
Acids Res., 33, 511–518.
2. Sander,C. and Schneider,R. (1991) Database of homology-derived
structures and the structurally meaning of sequence alignment.
Proteins, 9, 56–68.
3. Holm,L. and Sander,C. (1996) Mapping the protein universe. Science,
283, 595–602.
4. Lesk,A.M. and Chothia,C. (1980) How different amino acid
sequences determine similar protein structures: the structure
and evolutionary dynamics of the globins. J. Mol. Biol., 136,
225–270.
(a)
(b)
(c)
Figure 2. Computation of an Expresso Alignment. (a) Default T-Coffee alignment of the thioredoxin HOMSTRAD dataset. Red portions have a high reliability
and are expected to be more accurate that the rest. Blue and green portions are the less consistent. Consistency is estimated from a sequence-based T-Coffee library.
In this MSA 15% of the columns are similar to the reference HOMSTRAD MSA. (b) Expresso Alignment. Consistency is now estimated from a library computed
using template-based alignments. In this alignment 49% of the columns are similar to the HOMSTRAD reference MSA. (c) Automatic template assignment.
Nucleic Acids Research, 2006, Vol. 34, Web Server issue W607
5. Kouranov,A., Xie,L., de la Cruz,J., Chen,L., Westbrook,J.,
Bourne,P.E. and Berman,H.M. (2006) The RCSB PDB information
portal for structural genomics. Nucleic Acids Res., 34,
D302–D305.
6. Heringa,J. (1999) Two strategies for sequence comparison:
profile-preprocessed and secondary structure-induced multiple
alignment. Comput. Chem., 23, 341–364.
7. Simossis,V.A. and Heringa,J. (2005) PRALINE: a multiple sequence
alignment toolbox that integrates homology-extended and secondary
structure information. Nucleic Acids Res., 33, W289–W294.
8. Zhang,Z., Lindstam,M., Unge,J., Peterson,C. and Lu,G. (2003)
Potential for dramatic improvement in sequence alignment against
structures of remote homologous proteins by extracting structural
information from multiple structure alignment. J. Mol. Biol.,
332, 127–142.
9. Ren,T., Veeramalai,M., Tan,A.C. and Gilbert,D. (2004) MSAT: a
multiple sequence alignment tool based on TOPS. Appl. Bioinformatics,
3, 149–158.
10. Kleinjung,J., Romein,J., Lin,K. and Heringa,J. (2004) Contact-based
sequence alignment. Nucleic Acids Res., 32, 2464–2473.
11. O’Sullivan,O., Suhre,K., Abergel,C., Higgins,D.G. and Notredame,C.
(2004) 3DCoffee: combining protein sequences and structures
within multiple sequence alignments. J. Mol. Biol., 340, 385–395.
12. Notredame,C., Higgins,D.G. and Heringa,J. (2000) T-Coffee: a novel
method for fast and accurate multiple sequence alignment. J. Mol.
Biol., 302, 205–217.
13. Taylor,W.R. and Orengo,C.A. (1989) Protein structure alignment.
J. Mol. Biol., 208, 1–22.
14. Kleywegt,G.J. and Jones,T.A. (1999) Software for handling
macromolecular envelopes. Acta. Crystallogr. D Biol. Crystallogr.,
55, 941–944.
15. Kolodny,R., Koehl,P. and Levitt,M. (2005) Comprehensive evaluation of
protein structure alignment methods: scoring by geometric measures.
J. Mol. Biol., 346, 1173–1188.
16. Shi,J., Blundell,T.L. and Mizuguchi,K. (2001) FUGUE:
sequence-structure homology recognition using environment-specific
substitution tables and structure-dependent gap penalties. J. Mol.
Biol., 310, 243–257.
17. Poirot,O., Suhre,K., Abergel,C., O’Toole,E. and Notredame,C. (2004)
3DCoffee@igs: a web server for combining sequences and structures
into a multiple sequence alignment. Nucleic Acids Res., 32,
W37–W40.
18. de Bakker,P.I., Bateman,A., Burke,D.F., Miguel,R.N., Mizuguchi,K.,
Shi,J.,Shirai,H.andBlundell,T.L.(2001)HOMSTRAD:addingsequence
information to structure-based alignments of homologous protein
families. Bioinformatics, 17, 748–749.
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