the comparison of sequences of related proteins , which are diverse at the sequence level , can reveal features that are important for both structure and function .
the structural classification of proteins ( scop ) database ( 1,2 ) groups together protein structural domains in a hierarchical manner according to class , fold , superfamily and family .
importantly , relationships between proteins grouped at the superfamily level will often not be apparent from the consideration of sequence alone .
the astral compendium ( 3 ) provides subsets of scop clustered at various levels of sequence identity and is useful for selecting domains that are diverse at the sequence level .
s4 ( structure - based sequence alignments of scop superfamilies ) provides multiple structure - based alignments of scop ( version 1.63 ) protein superfamilies where no two domains share more than 40% sequence identity as defined by astral .
although some superfamilies are highly populated many have only one domain ( only the four main scop classes are considered in this paper , as they are the classes for which superfamily relationships can be considered meaningful ) .
despite this there are 456 superfamilies with more than one domain for which the database provides alignments . structural alignment and analysis of protein superfamilies
campass ( 5 ) is a database of structurally aligned protein superfamilies , available via the web , where no two proteins share a sequence identity more than 25% .
the homstrad database ( 4 ) is a valuable resource containing aligned three - dimensional structures of homologous proteins .
however , it is sometimes difficult to structurally align all the sequences in a family and homstrad may choose to split a group of proteins into two separate families , since the focus is to correctly align functionally and structurally important residues .
a similar database to ours is pass2 ( 6,7 ) , which is generated using comparer ( 8) from initial equivalences generated by stamp ( 9 ) or malign ( 10 ) .
assessing the accuracy of alignments generated by automated procedures is very difficult due to a lack of hand - crafted alignments of sequence diverse proteins . here , we show that our alignments are very similar to a handful of alignments produced manually by domain experts as evidence of the quality of the alignments .
we also show that the alignments correctly equivalence known sequence signatures corresponding to a functional binding site .
the multiple structure - based alignments are constructed using the program sap ( 11 ) for carrying out pairwise structural alignments and t - coffee ( 12 ) to perform a progressive hierarchical alignment using the information from the pairwise sap scores .
the t - coffee algorithm has the advantage that it allows information from all sequences to be considered at each alignment step in an hierarchical alignment procedure .
the t - coffee method has been used ( 13 ) for constructing alignments for functional families as part of the dali domain dictionary .
the method has also been shown to be successfully used for a mixture of sequence and structural information ( 14,15 ) .
the protocol we have adopted is listed in detail : 
 for each pair of domains a , b in a superfamily s : 
 use sap to generate a structural superposition of the domains , which generates a list of the three - dimensional co - ordinates x of the c atoms for each domain in the superposition , xi and xj , where i and j run over the lengths of the two domains ; a list of pairs of residues indices { i , j}l , l = 1 ,  ,
n , where the algorithm has equivalenced n residues ; and the overall rmsd of the alignment rmsdab.create a t - coffee library lab for the superposition of a and b.
the library has n entries , each entry specifies the equivalenced residues { i , j}l and the weight , wij that the t - coffee algorithm should give to the equivalencing of i and j. we calculate wij using the following formula : where k is constant.now generate a multiple sequence alignment of superfamily s by running t - coffee with all libraries lab with a  s , b  s and a  b. 
 we have experimented with various formulas for the weighting function for calculating w and found this method to give high - quality results .
the motivation behind the formula is that the certainty that the two residues are equivalent should be weighted by not only the global similarity of the two domains , but also how well the particular residues superpose .
we use a value of k = 1000 to generate the alignments . for each pair of domains a , b in a superfamily s : 
 use sap to generate a structural superposition of the domains , which generates a list of the three - dimensional co - ordinates x of the c atoms for each domain in the superposition , xi and xj , where i and j run over the lengths of the two domains ; a list of pairs of residues indices { i , j}l , l = 1 ,  , n , where the algorithm has equivalenced n residues ; and the overall rmsd of the alignment rmsdab.create a t - coffee library lab for the superposition of a and b.
the library has n entries , each entry specifies the equivalenced residues { i , j}l and the weight , wij that the t - coffee algorithm should give to the equivalencing of i and j. we calculate wij using the following formula : where k is constant .
use sap to generate a structural superposition of the domains , which generates a list of the three - dimensional co - ordinates x of the c atoms for each domain in the superposition , xi and xj , where i and j run over the lengths of the two domains ; a list of pairs of residues indices { i , j}l , l = 1 ,  ,
n , where the algorithm has equivalenced n residues ; and the overall rmsd of the alignment rmsdab . create a t - coffee library lab for the superposition of a and b. the library has n entries , each entry specifies the equivalenced residues { i , j}l and the weight , wij that the t - coffee algorithm should give to the equivalencing of i and j. we calculate wij using the following formula : now generate a multiple sequence alignment of superfamily s by running t - coffee with all libraries lab with a  s , b  s and a  b.
in the absence of hand - crafted multiple alignments constructed by domain experts , it is difficult to access the accuracy of multiple structure - based alignments .
( 16 ) . in their paper , three families are aligned : the long - chain four - helical cytokines , short - chain four - helical cytokines and the four - helical cytochromes . in aligning these families , hill et al . examined not only rmsds , but also hydrogen bonding , accessible surface areas and inter - residue contacts in producing pairwise alignments .
the four - helical cytokines have a unique up - up - down - down topology so far only observed in these cytokines .
they represent a difficult family for structural alignment , since they are structurally diverse showing an average rmsd between members of the superfamily of 3.12  ( from the sap alignments ) .
the scop superfamily has three families : long chain , short chain and interferon / interleukin 10 family .
we selected the proteins aligned in both our alignments and the reference alignments , and marked on the common cores that should be aligned .
the major error is the alignment of 1scf , which fails to align to any core region .
there are some more minor errors : part of the helix 3 of 1bgc has been misaligned , helix 2 of 1hul is also misaligned and most of the parts of the common core that are gaps are not correctly gapped . despite this the alignment appears to be of high quality .
we found that the four - helical cytochromes were aligned in a way that the core agreed completely with the reference alignment .
these data are summarized in table 1 , which shows alignment accuracies compared between pass2 ( database version august 20 , 2003 ) and s4 .
what is evident is that s4 alignments are closer to the reference alignments than pass2 in the three cases studied .
it is also important that an alignment correctly aligns the functionally important residues in a superfamily .
inspection of the s4 alignment shows that this signature is aligned across every member of the superfamily , in contrast to the pass2 alignment that fails to align this signature .
we are aware , however , that different protocols may perform better with certain types of superfamilies and our study only covers three reference alignments .
all the alignments in the s4 database can be downloaded as both clustal and fasta files , or the database can be browsed through our website .
the website provides additional markup to the downloadable alignments : the alignments can be displayed with either sequence or structure annotation .
the sequence annotation uses mview ( 17 ) to colour residues according to their physiochemical properties , allowing sequence conservation patterns to be seen .
the consensus group and median distance between equivalenced residues are also shown at each position ( for all residues equivalenced in a column of the multiple alignment , the distance between residues in all the pairwise alignments are calculated , and the median of the distances calculated ; the median is taken to lessen the effect of outliers from any poor pairwise structural alignments ) .
there is also a search facility into which a user can specify a given scop superfamily , scop domain name or pdb name .
our survey of existing resources revealed that they either only covered a subset of the available proteins in a superfamily that were structurally conserved or showed significant differences to reference alignments .
the use of a handful of reference alignments should not be considered a comprehensive assessment of the accuracy of the protocol we have used . in particular , our reference alignments are from the all alpha class  testing against alignments from other classes would be informative .
we have experimented with using profiles built from our structure - based alignments in profile  profile searching methods for detecting remote homologues and this approach shows some promise .
the alignments may be useful for protein modelling as an aid in aligning the sequence to be modelled with a collection of diverse templates .
the database generation procedure is automated and we therefore anticipate updating the database on a periodic basis to reflect newer releases of the scop database .
 s4 is an automatically generated database of multiple structure - based sequence alignments of protein superfamilies in the scop database . 
 all structural domains that do not share more than 40% sequence identity as defined by the astral compendium of protein structures are included . 
 the alignments are constructed using pairwise structural alignments to generate residue equivalences that are then integrated into multiple alignments using sequence alignment tools . 
 we describe the database and give examples showing how the automatically generated s4 alignments compare favourably to hand - crafted alignments . 
 available at : http://compbio.mds.qmw.ac.uk/s4.html . 
INTRODUCTION
PROTOCOL FOR CREATING ALIGNMENTS
QUALITY OF ALIGNMENTS
THE S4 DATABASE
SUMMARY