the quality of multiple sequence alignments directly affects their applications in structure modeling , similarity searches , function prediction and phylogenetic analysis . constructing accurate multiple alignments for distantly related proteins remains a difficult task in computational biology . aligning all sequences simultaneously by dynamic programing is not feasible for more than a few sequences ( 1 ) . therefore , many current programs use the heuristic progressive alignment technique , which reduces the problem of aligning multiple sequences to make a limited number of pairwise alignments . although progressive methods can be very fast , errors made at early stages are not corrected later . classic progressive methods based on general amino acid substitution matrices such as clustalw ( 2 ) can give reasonable results for similar sequences , but fail to produce accurate alignments for divergent sequences ( 3 ) . refinement after progressive steps can correct alignment errors , as implemented in recent programs such as mafft ( 4 ) and muscle ( 5 ) . the consistency - based alignment strategy ( 6 ) derives a better scoring function than general substitution matrices before carrying out the progressive alignment steps . using additional information from database homologs and known or predicted structures can lead to further improvement of alignment quality ( 4,710 ) . our progressive method promals ( 11 ) integrates advanced alignment techniques such as probabilistic consistency of profile  profile comparisons , and additional information from database homologs and predicted secondary structures . in promals3d ( 12 ) , alignment constraints from 3d structural comparisons are automatically derived and combined with constraints of promals profile  profile alignments with secondary structures to derive consistency - based alignments . promals3d has shown prominent improvements when 3d structures are available ( table 1 ) . 
 table 1.evaluation of alignment methods on sabmark and prefab benchmarksmethodsabmark - twi ( 209/7.7)sabmark - sup ( 425/8.3)prefab ( 1682/45.2)promals3d0.6160.8120.900promals0.3910.6650.790spem ( 10)0.3260.6280.774mummals ( 24)0.1960.5220.731probcons ( 16)0.1660.4850.716mafft0.1840.5100.722muscle0.1360.4330.680clustalw0.1270.3900.617average q - scores of two sabmark ( 25 ) data sets (  twi for  twilight zone set ,  sup for  superfamilies set ) and the prefab 4.0 ( 5 ) data set are shown . q - score is the number of correctly aligned residue pairs in the test alignment divided by the total number of aligned residue pairs in the reference alignment . for each data set , the two numbers in the parentheses separated by a slash are the number of alignments tested and the average number of sequences per alignment , respectively . for each data set , promals3d yields statistically higher accuracy than any other method ( p - value < 0.000001 ) according to wilcoxon signed rank test . evaluation of alignment methods on sabmark and prefab benchmarks average q - scores of two sabmark ( 25 ) data sets ( sup for  superfamilies set ) and the prefab 4.0 ( 5 ) data set are shown . q - score is the number of correctly aligned residue pairs in the test alignment divided by the total number of aligned residue pairs in the reference alignment . for each data set , the two numbers in the parentheses separated by a slash are the number of alignments tested and the average number of sequences per alignment , respectively . for each data set , promals3d yields statistically higher accuracy than any other method ( p - value < 0.000001 ) according to wilcoxon signed rank test . here we describe the promals3d web server that constructs alignments for multiple protein sequences and/or structures . the output is a consensus alignment that brings together sequence and structural information about input proteins and their homologs . promals3d server provides researchers a tool to produce high - quality alignments consistent with both sequences and structures in an automatic fashion . in addition to alignment construction , the server facilitates further analysis of target proteins by providing intermediate results of sequence and structural database searching , and presenting alignments with useful information about predicted secondary structures , sequence grouping and consensus sequences . promals3d ( 12 ) is a progressive method that clusters similar sequences and aligns them in a fast way , and uses more elaborate techniques to align the relatively divergent clusters to each other . in the first alignment stage , promals3d aligns similar sequences using a scoring function of weighted sum - of - pairs of blosum62 ( 13 ) scores . the first stage is fast and results in a number of prealigned groups ( clusters ) that are relatively distant from each other . in the second alignment stage representative sequences ( also called targets or target sequences below ) are subject to psi - blast searches for additional homologs from uniref90 ( 14 ) database and to psipred ( 15 ) secondary structure prediction . then a hidden markov model of profile  profile alignments with predicted secondary structure scoring is applied to pairs of representatives to derive sequence - based constraints . structure - based constraints are derived from homologs with known structures ( see details below ) and are combined with sequence - based constraints to derive a probabilistic consistency scoring function ( 16 ) . the representative sequences are progressively aligned using such a consistency scoring function , and the prealigned groups obtained in the first stage are merged into the alignment of representatives to form the final multiple sequence alignment . in promals3d , first , the program identifies homologs with 3d structures ( homolog3d ) for representative sequences . for each representative sequence , the profile of psi - blast ( stored as a checkpoint file ) search against the uniref90 database is used to initiate a new psi - blast search ( one iteration , with -c option ) against the scop40 domain database ( 17,18 ) that contains protein domain sequences with known structures . only structural domains that pass certain similarity criteria ( default : e - value < 0.001 and sequence identity no < 20% ) are kept . multiple homolog3ds could be identified and used for one target sequence if it contains several distinct domains with known structures . pairwise residue match constraints for two representative target sequences are derived from sequence - based target - to - homolog3d alignments and structure - based homolog3d - to - homolog3d alignments . for example , if residue a in target s1 is aligned to residue b in homolog3d t1 , residue b in homolog3d t1 is aligned with residue c in homolog3d t2 according to a structure comparison program , and residue c in homolog3d t2 is aligned with residue d in target s2 , then we deduce that residue a in sequence s1 is aligned with residue d in sequence s2 , and this pair ( a , d ) is used as a structure - derived constraint ( figure 1 ) . the alignment between a target sequence and its homolog3d can be the psi - blast alignment , or they can be re - aligned by the profile  profile comparison routine used in promals . the structure constraints among target sequences are combined with those constraints derived from profile  profile comparisons in the original promals to deduce a consistency - based scoring function that integrates database sequence profiles , predicted secondary structures and 3d structural information . we used an empirical weight ratio of 1.5 ( can be modified in server ) for structure constraints relative to the sequence constraints of profile  profile comparison in the original promals s1 and s2 are two target sequences . t1 and t2 are their homolog3ds . the alignment between two sequences s1 and s2 is deduced from two sequence - based sequence - to - homolog3d alignments and one structure - based homolog3d - to - homolog3d alignment . the three aligned residue pairs ( a , b ) , ( b , c ) and ( c , d ) indicate that the pair ( a , d ) is aligned in the deduced alignment between two targets . deducing alignment constraints using homologs with 3d structures ( homolog3ds ) . s1 and s2 are two target sequences . t1 and t2 are their homolog3ds . the alignment between two sequences s1 and s2 is deduced from two sequence - based sequence - to - homolog3d alignments and one structure - based homolog3d - to - homolog3d alignment . the three aligned residue pairs ( a , b ) , ( b , c ) and ( c , d ) indicate that the pair ( a , d ) is aligned in the deduced alignment between two targets . the promals3d web server is available at : http://prodata.swmed.edu / promals3d/. users can input or upload protein sequences , structures or user - defined alignment constraints . in addition to uploading bulky structural files , users can also specify just the pdb ids and chain ids . the output is a multiple alignment of input sequences and sequences extracted from input structures . it is also recommended that the user provides an email address to receive alignment results , as promals3d can take a considerable amount of time ( several hours ) to finish for a large number of divergent sequences , mainly due to the time - consuming steps of running psi - blast searches and calculating the profile - based consistency scoring function . one important parameter is the identity threshold that determines the partition of fast alignment stage and slow alignment stage , and thus balances alignment quality and speed . lowering this threshold can cause more sequences to be aligned in a fast but less accurate way , resulting in fewer representative groups subject to the time- and memory - consuming steps of psi - blast searches , structural comparisons and profile consistency measure . this tradeoff generally leads to less memory usage and computational time but potentially lower alignment quality . if the number of prealigned groups is large ( e.g. > 60 ) , promals3d could run out of memory during the consistency measure step . therefore , if the number of prealigned groups is above a threshold ( currently 60 ) , the server automatically adjusts the identity threshold to keep the number of prealigned groups to a fixed number ( currently 60 ) . we also provide options for changing weights of sequence - based constraints and structural - based constraints , and the weights of amino acid scoring and predicted secondary structure scoring in profile  profile alignments . parameters for running psi - blast and processing psi - blast alignments ( for generating amino acid profiles ) are also provided , such as e - value cutoff , the number of psi - blast iterations , identity cutoff to remove divergent hits and the number of homologs kept for profile calculation . for structure alignments of input structures or homologs with 3d structures , we provide options of using any combination of three structural comparison programs : dalilite ( 19 ) , fast ( 20 ) and tm - align ( 21 ) . three alignment formats can be accessed in this page : clustal format , fasta format and a colored alignment format . sequences in the alignment can be displayed in aligned order or input order . in a colored alignment , useful information about sequence grouping , secondary structure predictions , positional conservation and consensus sequences ( figure 2b ) is reported . sequence grouping is reflected by the color of sequence names if sequences are in aligned order . sequences with magenta names are representatives from prealigned groups . sequences with black names immediately under a representative sequence belong to the same prealigned group as the representative sequence . predicted secondary structures are shown for representative sequences ( residues with red and blue fonts are predicted to be -helices and -strands , respectively ) . above each alignment block , conserved positions are marked by their conservation indices ( integer values from 0 to 9 ) calculated using our program al2co ( 22 ) . the two lines beneath each alignment block show the consensus amino acid sequence ( with symbols explained in figure 2 legend ) and consensus secondary structure predictions (  h : -helix ;  e : -strand ) . such a coloring and labeling scheme is helpful for further sequence and structural analysis of input sequences and structures . in addition to the alignments , the server also provides links to the original input sequences and structures and intermediate results such as the guide tree , psi - blast alignments , detected homologs with 3d structures , psipred secondary structure predictions . this alignment is for input of four sh2 domain sequences selected from pfam database ( 23 ) and four sh2 structures ( pdb ids are 1aya , 1jyr , 1lkk and 1mil ) . the first line in each alignment block begins with  conservation: and shows conservation index numbers for conserved positions . consensus_ss: shows the consensus secondary structure predictions (  h : -helix ;  e : -strand ) . if the weighted frequency of certain type of residues is above a certain threshold , the consensus symbol of that type is displayed . symbols are provided for the following types : conserved amino acid residues : bold and uppercase letters ; aliphatic residues ( i , v , l ) : l ; aromatic residues ( y , h , w , f ) : @ ; hydrophobic residues ( w , f , y , m , l , i , v , a , c , t , h ) : h ; alcohol residues ( s , t ) : o ; polar residues ( d , e , h , k , n , q , r , s , t ) : p ; tiny residues ( a , g , c , s ) : t ; small residues ( a , g , c , s , v , n , d , t , p ) : s ; bulky residues ( e , f , i , k , l , m , q , r , w , y ) : b ; positively charged residues ( k , r , h ) : + ; negatively charged residues ( d , e ) :  ; charged ( d , e , k , r , h ) : c. each representative sequence has a magenta name and is colored according to psipred secondary structure predictions ( red : -helix , blue : -strand ) . a representative sequence and the immediate sequences below it with black names , if there are any , form a closely related group and they are aligned in the first stage . ( a ) output page of the promals3d server . this alignment is for input of four sh2 domain sequences selected from pfam database ( 23 ) and four sh2 structures ( pdb ids are 1aya , 1jyr , 1lkk and 1mil ) . the first line in each alignment block begins with  conservation: and shows conservation index numbers for conserved positions . consensus_ss: shows the consensus secondary structure predictions (  h : -helix ;  e : -strand ) . if the weighted frequency of certain type of residues is above a certain threshold , the consensus symbol of that type is displayed . symbols are provided for the following types : conserved amino acid residues : bold and uppercase letters ; aliphatic residues ( i , v , l ) : l ; aromatic residues ( y , h , w , f ) : @ ; hydrophobic residues ( w , f , y , m , l , i , v , a , c , t , h ) : h ; alcohol residues ( s , t ) : o ; polar residues ( d , e , h , k , n , q , r , s , t ) : p ; tiny residues ( a , g , c , s ) : t ; small residues ( a , g , c , s , v , n , d , t , p ) : s ; bulky residues ( e , f , i , k , l , m , q , r , w , y ) : b ; positively charged residues ( k , r , h ) : + ; negatively charged residues ( d , e ) :  ; charged ( d , e , k , r , h ) : c. each representative sequence has a magenta name and is colored according to psipred secondary structure predictions ( red : -helix , blue : -strand ) . a representative sequence and the immediate sequences below it with black names , if there are any , form a closely related group and they are aligned in the first stage . users can input or upload protein sequences , structures or user - defined alignment constraints . in addition to uploading bulky structural files , users can also specify just the pdb ids and chain ids . the output is a multiple alignment of input sequences and sequences extracted from input structures . it is also recommended that the user provides an email address to receive alignment results , as promals3d can take a considerable amount of time ( several hours ) to finish for a large number of divergent sequences , mainly due to the time - consuming steps of running psi - blast searches and calculating the profile - based consistency scoring function . one important parameter is the identity threshold that determines the partition of fast alignment stage and slow alignment stage , and thus balances alignment quality and speed . lowering this threshold can cause more sequences to be aligned in a fast but less accurate way , resulting in fewer representative groups subject to the time- and memory - consuming steps of psi - blast searches , structural comparisons and profile consistency measure . this tradeoff generally leads to less memory usage and computational time but potentially lower alignment quality . if the number of prealigned groups is large ( e.g. > 60 ) , promals3d could run out of memory during the consistency measure step . therefore , if the number of prealigned groups is above a threshold ( currently 60 ) , the server automatically adjusts the identity threshold to keep the number of prealigned groups to a fixed number ( currently 60 ) . we also provide options for changing weights of sequence - based constraints and structural - based constraints , and the weights of amino acid scoring and predicted secondary structure scoring in profile  profile alignments . parameters for running psi - blast and processing psi - blast alignments ( for generating amino acid profiles ) are also provided , such as e - value cutoff , the number of psi - blast iterations , identity cutoff to remove divergent hits and the number of homologs kept for profile calculation . for structure alignments of input structures or homologs with 3d structures , we provide options of using any combination of three structural comparison programs : dalilite ( 19 ) , fast ( 20 ) and tm - align ( 21 ) . three alignment formats can be accessed in this page : clustal format , fasta format and a colored alignment format . sequences in the alignment can be displayed in aligned order or input order . in a colored alignment , useful information about sequence grouping , secondary structure predictions , positional conservation and consensus sequences ( figure 2b ) is reported . sequence grouping is reflected by the color of sequence names if sequences are in aligned order . sequences with black names immediately under a representative sequence belong to the same prealigned group as the representative sequence . predicted secondary structures are shown for representative sequences ( residues with red and blue fonts are predicted to be -helices and -strands , respectively ) . above each alignment block , conserved positions are marked by their conservation indices ( integer values from 0 to 9 ) calculated using our program al2co ( 22 ) . the two lines beneath each alignment block show the consensus amino acid sequence ( with symbols explained in figure 2 legend ) and consensus secondary structure predictions (  h : -helix ;  e : -strand ) . such a coloring and labeling scheme is helpful for further sequence and structural analysis of input sequences and structures . in addition to the alignments , the server also provides links to the original input sequences and structures and intermediate results such as the guide tree , psi - blast alignments , detected homologs with 3d structures , psipred secondary structure predictions . this alignment is for input of four sh2 domain sequences selected from pfam database ( 23 ) and four sh2 structures ( pdb ids are 1aya , 1jyr , 1lkk and 1mil ) . the first line in each alignment block begins with  conservation: and shows conservation index numbers for conserved positions . consensus_ss: shows the consensus secondary structure predictions (  h : -helix ;  e : -strand ) . if the weighted frequency of certain type of residues is above a certain threshold , the consensus symbol of that type is displayed . symbols are provided for the following types : conserved amino acid residues : bold and uppercase letters ; aliphatic residues ( i , v , l ) : l ; aromatic residues ( y , h , w , f ) : @ ; hydrophobic residues ( w , f , y , m , l , i , v , a , c , t , h ) : h ; alcohol residues ( s , t ) : o ; polar residues ( d , e , h , k , n , q , r , s , t ) : p ; tiny residues ( a , g , c , s ) : t ; small residues ( a , g , c , s , v , n , d , t , p ) : s ; bulky residues ( e , f , i , k , l , m , q , r , w , y ) : b ; positively charged residues ( k , r , h ) : + ; negatively charged residues ( d , e ) :  ; charged ( d , e , k , r , h ) : c. each representative sequence has a magenta name and is colored according to psipred secondary structure predictions ( red : -helix , blue : -strand ) . a representative sequence and the immediate sequences below it with black names , if there are any , form a closely related group and they are aligned in the first stage . ( a ) output page of the promals3d server . this alignment is for input of four sh2 domain sequences selected from pfam database ( 23 ) and four sh2 structures ( pdb ids are 1aya , 1jyr , 1lkk and 1mil ) . the first line in each alignment block begins with  conservation: and shows conservation index numbers for conserved positions . consensus_ss: shows the consensus secondary structure predictions (  h : -helix ;  e : -strand ) . if the weighted frequency of certain type of residues is above a certain threshold , the consensus symbol of that type is displayed . symbols are provided for the following types : conserved amino acid residues : bold and uppercase letters ; aliphatic residues ( i , v , l ) : l ; aromatic residues ( y , h , w , f ) : @ ; hydrophobic residues ( w , f , y , m , l , i , v , a , c , t , h ) : h ; alcohol residues ( s , t ) : o ; polar residues ( d , e , h , k , n , q , r , s , t ) : p ; tiny residues ( a , g , c , s ) : t ; small residues ( a , g , c , s , v , n , d , t , p ) : s ; bulky residues ( e , f , i , k , l , m , q , r , w , y ) : b ; positively charged residues ( k , r , h ) : + ; negatively charged residues ( d , e ) :  ; charged ( d , e , k , r , h ) : c. each representative sequence has a magenta name and is colored according to psipred secondary structure predictions ( red : -helix , blue : -strand ) . a representative sequence and the immediate sequences below it with black names , if there are any , form a closely related group and they are aligned in the first stage .
multiple sequence alignments are essential in computational sequence and structural analysis , with applications in homology detection , structure modeling , function prediction and phylogenetic analysis . 
 we report promals3d web server for constructing alignments for multiple protein sequences and/or structures using information from available 3d structures , database homologs and predicted secondary structures . 
 promals3d shows higher alignment accuracy than a number of other advanced methods . 
 input of promals3d web server can be fasta format protein sequences , pdb format protein structures and/or user - defined alignment constraints . 
 the output page provides alignments with several formats , including a colored alignment augmented with useful information about sequence grouping , predicted secondary structures and consensus sequences . 
 intermediate results of sequence and structural database searches are also available . 
 the promals3d web server is available at : http://prodata.swmed.edu / promals3d/.