for many purposes , it is useful to have a list of protein sequences of known structure from entries in the protein data bank ( pdb ) ( 1 ) that fit certain user - defined criteria .
these purposes include statistical analysis of certain structural features , such as side - chain rotamer distributions ( 2 ) , benchmarking sequence alignment and structure prediction methods ( 3 ) , and analysis of a set of proteins , such as those with a certain function ( e.g. dna - binding ) ( 4 ) .
the criteria for limiting the list of sequences can be applied to each chain in a pdb file , to each entry as a whole or to the relationships between protein chains and entries .
chain - specific criteria include function , species , sequence length and whether the chain is presented with only c atoms in the pdb file or all atoms .
entry - specific criteria include the function of a complex , resolution , r - factor , experiment type ( nmr or x - ray ) and other structure quality criteria .
there are a number of servers that provide such lists , including the reprdb ( 5 ) , astral ( 6 ) , uniqueprot ( 7 ) sites and the pdb site itself . each provides a variety of services for specific purposes .
they vary in a number of respects , including how sequence identities are determined and the flexibility and extent of services available to the user . in this paper , we describe extensions to our pisces server that provides some advantages over other such servers , depending on the purposes of the user .
first , we believe in general that local alignments are better than global alignments for the purpose of selecting sequences from the pdb dataset , since many proteins share homology in one domain that may represent only a portion of the complete sequence .
servers that use clustal w ( 8) , which performs global alignments , may underestimate sequence identity for shared domains by aligning over unrelated portions of two sequences .
it is also important that sequence alignments cover the full - length of related regions of each protein pair .
blast [ and to a lesser extent psi - blast ( 9 ) ] may overestimate sequence identity for distantly related proteins by providing incomplete alignments over only the most - conserved regions of the sequence pair . overestimating sequence identities results in lists that are shorter than necessary since
structure - based alignments are likely to provide the most complete and accurate alignments at low - sequence identity than any sequence - based alignment method ( 10 ) . as described below , pisces now uses a combination of structure alignments at low - sequence identity and sequence alignment using psi - blast at high - sequence identity . other criteria than strict sequence identity
second , sequence culling servers may provide services for a variety of purposes . [ the verb to cull can be used in different senses .
the original meaning ( webster 's 1913 ) was to select or gather , as in flowers .
more recently , the word is used in two ways : culling the weakest members of a herd , or culling the herd itself , leaving behind the healthiest animals .
we are generally using it in the second sense , to cull the pdb by removing redundant or low - resolution structures . ] in addition to culling the entire pdb , a user may wish to cull a subset of the pdb .
for instance , a user may have a list of pdb entries that contain antibody structures and may want a subset of these with certain structural quality and mutual sequence identity .
one can also apply the sequence identity  by - entry rather than  by - chain. we define this as removing a pdb entry ( that may contain more than one chain ) from the list if another entry already in the list has at least one chain that has a sequence identity , higher than the cut - off , to any chain in the proposed entry
. this might be useful if one wants a list of unique complexes in the pdb .
another aspect of such a server is the kind of annotation provided in the results .
each server returns a list of chains and some servers are able to return a list of fasta - formatted sequences . usually , the annotation is simply the chain i d and sometimes what the rcsb provides in its 
however , the rcsb does not provide consistent annotation for each chain in each pdb file with multiple chains .
chain b is 2-microglobulin . in the fasta - formatted sequence database from the pdb ,
these two chains are given as : 
 2hla_a mol : protein length:270 human class i histocompatibility antigen aw2hla_b mol : protein length:99 human class i histocompatibility antigen aw 6 
 in fact , the lines are unfortunately truncated , but even the annotation from the  sequence details resource for the entry 2hla on rcsb 's site has only this for chain b :  human class i histocompatibility antigen aw 68.1. there is , however , more information in the full mmcif file , including the name 
beta-2-microglobulin as well as the swiss - prot entry accession number from which additional information may be obtained .
2hla_a mol : protein length:270 human class i histocompatibility antigen aw 2hla_b mol : protein length:99 human class i histocompatibility antigen aw 6 our pisces server was developed from a previous version referred to as 
culledpdb used blast to determine sequence identities , while pisces has used psi - blast to determine sequence identities ( 11 ) by building a position - specific scoring matrix or profile for each unique sequence in the pdb from a multi - round search of the non - redundant protein sequence database ( 12 ) .
pisces also provides a resource for culling a user - input list of chains or entries from the pdb or from a set of non - pdb sequences provided as either a list of genbank ids or in the fasta format .
we have shown that the use of the psi - blast profiles resulted in much longer lists than while using blast ( 11 ) , while not exhibiting errors associated with using a global alignment algorithm , such as clustal w. we have made several important improvements in pisces that are described in this paper : 
 we now use the structure alignment program ce ( 13 ) to refine the relationship determination between proteins and recalculate their sequence identities that are found by using psi-blast.pisces now use ce - type sequence identity , i.e. identical pairs divided by all aligned pairs excluding gaps .
psi - blast , in contrast , calculates the sequence identity by the ratio of the number of identical pairs to the full - length of the alignment , including gaps .
this change means that if two closely related proteins are aligned , but one has a large insertion , the sequence identity will remain high .
this may occur for instance if a long disordered loop is engineered out of a protein to facilitate crystallization .
the new definition of sequence identity is a more conservative criterion for sequence culling , since sequence identities are higher than they were previously using the psi - blast values , and more sequences are removed for a target sequence identity.pisces is now able to cull by - entry in addition to by - chain , as described above.pisces now provides more annotation for each chain in the pdb than the pdb itself does .
this annotation includes chain - specific gene names and functional and species information retrieved from the full mmcif files of the pdb , and the swiss - prot and genbank databases.from the re - engineered search engine of the pdb ( now in beta release , ) ( 14 ) , with a single click on the external sitespisces menu on the pdb 's search result page , all the hits or a selected subset of the results of a search can be transmitted to the pisces site for culling using a user 's input criteria for structural quality and sequence identity cut - offs .
we now use the structure alignment program ce ( 13 ) to refine the relationship determination between proteins and recalculate their sequence identities that are found by using psi - blast .
pisces now use ce - type sequence identity , i.e. identical pairs divided by all aligned pairs excluding gaps .
psi - blast , in contrast , calculates the sequence identity by the ratio of the number of identical pairs to the full - length of the alignment , including gaps .
this change means that if two closely related proteins are aligned , but one has a large insertion , the sequence identity will remain high .
this may occur for instance if a long disordered loop is engineered out of a protein to facilitate crystallization .
the new definition of sequence identity is a more conservative criterion for sequence culling , since sequence identities are higher than they were previously using the psi - blast values , and more sequences are removed for a target sequence identity .
pisces is now able to cull by - entry in addition to by - chain , as described above .
pisces now provides more annotation for each chain in the pdb than the pdb itself does .
this annotation includes chain - specific gene names and functional and species information retrieved from the full mmcif files of the pdb , and the swiss - prot and genbank databases . from the re - engineered search engine of the pdb ( now in beta release , ) ( 14 ) , with a single click on the external sitespisces menu on the pdb 's search result page , all the hits or a selected subset of the results of a search can be transmitted to the pisces site for culling using a user 's input criteria for structural quality and sequence identity cut - offs .
pisces uses the mmcif - format files from rcsb to determine sequences , experiment type , resolution , r - factors and other features of pdb entries and chains .
these mmcif files are a result of the uniformity project , an effort by the rcsb to standardize and correct information across all the pdb files ( 15,16 ) .
some missing values for resolution and r - factors are obtained from the pdbfinder database ( 17 ) .
pisces works from a fasta - formatted database of all sequences in the pdb called pdbaa , which is distinct from ncbi 's database of the pdb sequences with the same name ( ) .
our pdbaa database is available to users who want a complete set of sequences in the pdb .
it is also used to provide the sequences and annotations returned by pisces for subsets of the pdb .
pdbaa provides basic information on chain length , experiment type ( x - ray , nmr , etc . ) , resolution , r - factor and free r - factor as appropriate .
these pieces of information are useful if a user wants to use a structure as a template for homology modeling , and wants to choose the best template by searching the whole pdb . in order to integrate as much useful information as possible into the output of pisces
the goal is to have gene names for each sequence , swiss - prot or other database identifiers and species information .
these are obtained from the following sources : the mmcif files themselves , swiss - prot 's index of pdb entries ( pdbtosp.txt , ) , the protein identification resource ( pir ) and ncbi 's non - redundant protein sequence database , in that order to obtain the desired information .
our goal in culling the pdb is to provide the longest list possible of the highest resolution structures that fulfill the sequence identity and structural quality cut - offs . for this purpose , we need to identify potential evolutionary relationships and complete sequence alignment between related regions of each identified pair . historically , 98% of requests to pisces have been for culled lists at 25% identity or higher .
we have determined that psi - blast identifies 99.9% of such relationships with e - value of 1.0 or better ( data not shown ) , and thus we do not use structure alignment to identify evolutionary relationships .
rather , we use the structure alignment program ce ( 13 ) to align structure pairs which psi - blast determines as having a sequence identity of 50% or less or for which the psi - blast alignment covers < 80% of the shorter sequence in each pair .
we have found some cases where structure alignment sequence identities are much lower than those calculated with psi - blast even though the alignment lengths are comparable .
this may occur when one single - domain protein is homologous to each of the two domains in another protein .
ce may align the first protein to either of the two domains of the second protein , but not necessarily the more closely related one . to account for this
, we use the sequence identity obtained by either psi - blast or ce , whichever has the larger number of identically aligned residue pairs . with the sequence identities in hand
, pisces uses the method of hobohm and sander ( 18,19 ) to cull the sequences that pass the chain and entry criteria input by the user .
culling by - chain means treating each chain in each pdb entry as a separate entity .
based on the requests from a number of users of pisces , we have added another functionality to pisces : culling  by - entry. for this procedure , the sequence identity between any two entries is defined as the highest sequence identity of any one chain in one entry with any one chain in the other entry . in this way
, no two entries will appear in the same list if they share chains with sequence identity over the cut - off .
pisces further allows the user to choose whether to cull within each entry and allows the user to use another sequence identity cut - off for this culling procedure .
so for instance , if an entry is a homodimer , the user can choose to have both sequences returned ( no culling within entry ) or just one of them ( culling within entry by some value < 100% ) .
pisces first gives the user the option to : ( i ) cull the entire pdb ; ( ii ) cull from a search at the pdb 's re - engineered search engine site  this option takes the user to the pdb 's web page ( ) before returning to pisces ; ( iii ) cull from a user - input list of chains or entries ; ( iv ) cull from a list of genbank entries ; and ( v ) cull from a fasta - formatted set of sequences or from blast or psi - blast output . for options ( iv ) and ( v ) , sequence alignments
are obtained by using psi - blast on the list of input sequences to determine sequence identities .
it is assumed that these sequences are not in the pdb and , therefore , structural criteria are not used .
option ( i ) takes a user directly to a page for inputting structural quality criteria ( experiment type , resolution , r - factor , c-status , chain lengths , etc . ) and sequence identity cut - offs . option ( ii )
take a user to the rcsb site or the user may start at the rcsb site directly .
once the rcsb server has returned a list of entries that satisfy search parameters , the user can click on the external sitespisces menu on the rcsb page to return to pisces and to a page displaying the list of hits ( all or selected ) from rcsb .
option ( iii ) takes a user to a page for inputting a list of pdb entries or chains .
after the list of pdb entries or chains to be searched is confirmed by the user for options ( ii ) and ( iii ) , he / she is asked for the structural criteria to be used for culling .
pisces then confirms the input data and asks for user name , institution and email address .
when the results are ready , almost always within minutes , the server sends an email to the user to download the results from a page given in the email .
the results include a list of the input sequences ( if used ) , the input cut - offs , the list of chains or entries that result from culling and a fasta - formatted file of the selected sequences .
in addition to the sequence culling service , pisces also provides databases and programs that may prove useful to the user : 
 a fully annotated pdbaa database . for each pdb chain ,
the description line includes chain length , resolution and r - value ( for x - ray structures ) , protein descriptions , database name and entry name in the reference database , and species.two more condensed variants of pdbaa : pdbaa.ent and pdbaa.nr.pdbaa.ent has all redundant chains within entries removed and the removed chain ids are placed at the end of the description line of the representative chains .
pdbaa.nr is similar to pdbaa.ent , but it removes all redundant chains within the whole pdbaa instead of only within entries.a standalone package for pisces .
the standalone version of pisces has all the major features of the web - based pisces server . a fully annotated pdbaa database . for each pdb chain ,
the description line includes chain length , resolution and r - value ( for x - ray structures ) , protein descriptions , database name and entry name in the reference database , and species .
two more condensed variants of pdbaa : pdbaa.ent and pdbaa.nr.pdbaa.ent has all redundant chains within entries removed and the removed chain ids are placed at the end of the description line of the representative chains .
pdbaa.nr is similar to pdbaa.ent , but it removes all redundant chains within the whole pdbaa instead of only within entries . a standalone package for pisces .
the standalone version of pisces has all the major features of the web - based pisces server .
pisces uses the mmcif - format files from rcsb to determine sequences , experiment type , resolution , r - factors and other features of pdb entries and chains .
these mmcif files are a result of the uniformity project , an effort by the rcsb to standardize and correct information across all the pdb files ( 15,16 ) .
some missing values for resolution and r - factors are obtained from the pdbfinder database ( 17 ) .
pisces works from a fasta - formatted database of all sequences in the pdb called pdbaa , which is distinct from ncbi 's database of the pdb sequences with the same name ( ) .
our pdbaa database is available to users who want a complete set of sequences in the pdb .
it is also used to provide the sequences and annotations returned by pisces for subsets of the pdb .
pdbaa provides basic information on chain length , experiment type ( x - ray , nmr , etc . ) , resolution , r - factor and free r - factor as appropriate .
these pieces of information are useful if a user wants to use a structure as a template for homology modeling , and wants to choose the best template by searching the whole pdb . in order to integrate as much useful information as possible into the output of pisces
the goal is to have gene names for each sequence , swiss - prot or other database identifiers and species information .
these are obtained from the following sources : the mmcif files themselves , swiss - prot 's index of pdb entries ( pdbtosp.txt , ) , the protein identification resource ( pir ) and ncbi 's non - redundant protein sequence database , in that order to obtain the desired information .
our goal in culling the pdb is to provide the longest list possible of the highest resolution structures that fulfill the sequence identity and structural quality cut - offs . for this purpose
, we need to identify potential evolutionary relationships and complete sequence alignment between related regions of each identified pair .
historically , 98% of requests to pisces have been for culled lists at 25% identity or higher .
we have determined that psi - blast identifies 99.9% of such relationships with e - value of 1.0 or better ( data not shown ) , and thus we do not use structure alignment to identify evolutionary relationships . rather , we use the structure alignment program ce ( 13 ) to align structure pairs which psi - blast determines as having a sequence identity of 50% or less or for which the psi - blast alignment covers < 80% of the shorter sequence in each pair .
we have found some cases where structure alignment sequence identities are much lower than those calculated with psi - blast even though the alignment lengths are comparable .
this may occur when one single - domain protein is homologous to each of the two domains in another protein .
ce may align the first protein to either of the two domains of the second protein , but not necessarily the more closely related one . to account for this
, we use the sequence identity obtained by either psi - blast or ce , whichever has the larger number of identically aligned residue pairs .
with the sequence identities in hand , pisces uses the method of hobohm and sander ( 18,19 ) to cull the sequences that pass the chain and entry criteria input by the user .
culling by - chain means treating each chain in each pdb entry as a separate entity .
based on the requests from a number of users of pisces , we have added another functionality to pisces : culling  by - entry. for this procedure , the sequence identity between any two entries is defined as the highest sequence identity of any one chain in one entry with any one chain in the other entry . in this way
, no two entries will appear in the same list if they share chains with sequence identity over the cut - off .
pisces further allows the user to choose whether to cull within each entry and allows the user to use another sequence identity cut - off for this culling procedure .
so for instance , if an entry is a homodimer , the user can choose to have both sequences returned ( no culling within entry ) or just one of them ( culling within entry by some value < 100% ) .
pisces first gives the user the option to : ( i ) cull the entire pdb ; ( ii ) cull from a search at the pdb 's re - engineered search engine site  this option takes the user to the pdb 's web page ( ) before returning to pisces ; ( iii ) cull from a user - input list of chains or entries ; ( iv ) cull from a list of genbank entries ; and ( v ) cull from a fasta - formatted set of sequences or from blast or psi - blast output . for options ( iv ) and ( v ) ,
sequence alignments are obtained by using psi - blast on the list of input sequences to determine sequence identities .
it is assumed that these sequences are not in the pdb and , therefore , structural criteria are not used .
option ( i ) takes a user directly to a page for inputting structural quality criteria ( experiment type , resolution , r - factor , c-status , chain lengths , etc . ) and sequence identity cut - offs . option ( ii )
take a user to the rcsb site or the user may start at the rcsb site directly .
once the rcsb server has returned a list of entries that satisfy search parameters , the user can click on the external sitespisces menu on the rcsb page to return to pisces and to a page displaying the list of hits ( all or selected ) from rcsb . option ( iii )
takes a user to a page for inputting a list of pdb entries or chains . after the list of pdb entries or chains to be searched
is confirmed by the user for options ( ii ) and ( iii ) , he / she is asked for the structural criteria to be used for culling .
pisces then confirms the input data and asks for user name , institution and email address .
when the results are ready , almost always within minutes , the server sends an email to the user to download the results from a page given in the email .
the results include a list of the input sequences ( if used ) , the input cut - offs , the list of chains or entries that result from culling and a fasta - formatted file of the selected sequences .
in addition to the sequence culling service , pisces also provides databases and programs that may prove useful to the user : 
 a fully annotated pdbaa database . for each pdb chain ,
the description line includes chain length , resolution and r - value ( for x - ray structures ) , protein descriptions , database name and entry name in the reference database , and species.two more condensed variants of pdbaa : pdbaa.ent and pdbaa.nr.pdbaa.ent has all redundant chains within entries removed and the removed chain ids are placed at the end of the description line of the representative chains .
pdbaa.nr is similar to pdbaa.ent , but it removes all redundant chains within the whole pdbaa instead of only within entries.a standalone package for pisces .
the standalone version of pisces has all the major features of the web - based pisces server . a fully annotated pdbaa database .
for each pdb chain , the description line includes chain length , resolution and r - value ( for x - ray structures ) , protein descriptions , database name and entry name in the reference database , and species .
two more condensed variants of pdbaa : pdbaa.ent and pdbaa.nr.pdbaa.ent has all redundant chains within entries removed and the removed chain ids are placed at the end of the description line of the representative chains .
pdbaa.nr is similar to pdbaa.ent , but it removes all redundant chains within the whole pdbaa instead of only within entries .
the standalone version of pisces has all the major features of the web - based pisces server .
we investigated the effects of using structure - based alignments on the results of pisces . on the pdb dataset ,
the number of related pairs was : 637 115 according to blast ( e - value better than 1.0 , alignment length > 30 ) and 758 411 according to psi - blast ( e - value better than 1.0 , alignment length > 30 ) . in figure 1 , we have plotted sequence identities and alignment lengths for psi - blast versus blast and ce versus psi - blast .
the bottom two plots show that the psi - blast alignments are generally longer than the blast alignments , and the ce alignments are longer than the psi - blast alignments .
the top two plots show that the sequence identities are lower in psi - blast than in blast and lower in ce than in psi - blast .
as noted earlier , this is due to the fact that incomplete alignments result in artificially higher sequence identities , because the alignment occurs over the most - conserved regions of the two sequences .
we have boxed a region of the top two plots to show pairwise relationships that would be used to cull at 30% sequence identity .
for instance , in the upper right figure these points would be used to cull using psi - blast - determined sequence identities but not ce - determined identities , since the points in the box have psi - blast sequence identity > 30% but ce sequence identity < 30% .
there are far more points in this box than in the corresponding rectangular box for inclusion by psi - blast but not by ce ( to the left and above the green box ) . in table 1
, we show the lengths of lists under various sequence identity cut - offs as determined by using blast , psi - blast and the reprdb server ( 5 ) , which use clustal w and structure alignments .
ce within pisces provides longer lists at low - sequence identity than the sequence - based methods of blast and psi - blast . at very high - sequence identity ,
the results are based primarily on psi - blast calculations and thus are very similar . actually , the difference between ce and psi - blast is not only the list length , but also the content in the lists .
for example , for lists with 25% sequence identity cut - off , the ce list has only 57 more chains than the psi - blast list , but in fact the two lists have 157 different chains that are not in common .
its lists are longer at high - sequence identity , probably because the global alignment algorithm produces alignments over non - homologous regions of sequence pairs , thus lowering the sequence identity compared with a local alignment algorithm .
as shown in introduction , chain - specific information is sometimes missing in the fasta - formatted database available from the rcsb . for 2hla
, our pdbaa provides these annotations : 
 2hlaa 270 xray 2.60 0.173 na class i histocompatibility antigen ( hla - aw68 ) < sws 1a68_human > [ homo sapiens]2hlab 99 xray 2.60 0.173 na beta 2-microglobulin < sws b2mg_human > [ homo sapiens ] 
 after the chain identifier is the sequence length , experiment type , resolution , r - factor , free r - factor ( not available in this case ) , the gene name , the database reference in angular brackets ( in this case , swiss - prot entry b2mg_human ) and the species in square brackets .
while this information is available in the mmcif file for 2hla , it is not available in the fasta sequence database provided by the rcsb .
2hlaa 270 xray 2.60 0.173 na class i histocompatibility antigen ( hla - aw68 ) < sws 1a68_human > [ homo sapiens ] 2hlab 99 xray 2.60 0.173 na beta 2-microglobulin < sws b2mg_human
> [ homo sapiens ] finally , we expect the link to pdb 's search capabilities to be very useful . since the establishment of this link , almost 10% of the requests to pisces have come via the pdb search page .
the re - engineered pdb page is only in beta test phase , and so we expect that pisces will find additional utility when rcsb releases the final version of their search site .
scatterplots of sequence identities ( top figures ) and alignment lengths ( bottom figures ) for alignment pairs in common between psi - blast and blast ( left two figures ) and ce and psi - blast ( right two figures ) . on the top two plots , a box is shown that contains points that would be used to eliminate sequences at 30% sequence identity using the method on the x - axis but not by the method on the y - axis .
lengths of lists obtained using different sequence alignment methods criteria for inclusion in the lists : resolution 3.0  ; including c chains ; excluding non - x - ray entries .
 pisces is a database server for producing lists of sequences from the protein data bank ( pdb ) using a number of entry- and chain - specific criteria and mutual sequence identity . 
 our goal in culling the pdb is to provide the longest list possible of the highest resolution structures that fulfill the sequence identity and structural quality cut - offs . 
 the new pisces server uses a combination of psi - blast and structure - based alignments to determine sequence identities . 
 structure alignment produces more complete alignments and therefore more accurate sequence identities than psi - blast . 
 pisces now allows a user to cull the pdb by - entry in addition to the standard culling by individual chains . in this scenario 
 , a list will contain only entries that do not have a chain that has a sequence identity to any chain in any other entry in the list over the sequence identity cut - off . 
 pisces also provides fully annotated sequences including gene name and species . 
 the server allows a user to cull an input list of entries or chains , so that other criteria , such as function , can be used . 
 results from a search on the re - engineered rcsb 's site for the pdb can be entered into the pisces server by a single click , combining the powerful searching abilities of the pdb with pisces 's utilities for sequence culling . 
 the server 's data are updated weekly . 
 the server is available at . 
INTRODUCTION
METHODS
Database information and sequence annotation
Sequence identities
Culling by-chain and by-entry
Usage
RESULTS AND DISCUSSION
Figures and Tables