many proteins function as homo- and heterooligomers , but in most cases the size and actual structures of these multimers are not known from direct solution experiments such as analytical ultracentrifugation and nuclear magnetic resonance ( nmr ) .
instead , they are based only on what is observed in x - ray crystal structures , sometimes even a single crystal structure .
both the protein data bank ( pdb ) ( 1 ) and the european bioinformatics institute ( ebi ) ( 2 ) provide data on 
the pdb s author - defined biological units are based on what authors believe to be the biologically relevant structures , while the protein interfaces , surfaces and assemblies ( pisa ) ( 2 ) server from the ebi contains predicted oligomeric structures based on chemical thermodynamic calculations of complex stability .
in addition , the ebi provides the protein quaternary server ( pqs ) , although this site is no longer updated ( 3 ) .
the pins database at oak ridge national laboratory also contains predicted biological assemblies for structures in the pdb , but only those released prior to mid-2007 ( 4 ) .
many online databases have used the pdb , pqs and pisa biological assemblies to examine the interfaces between proteins , including pibase ( 5 ) , psimap / psibase ( 6 ) , snappi - db ( 7 ) , scoppi ( 8) , print ( 9 ) and ipfam ( 10 ) .
these sites present snapshots of the pdb at the time they were developed , and are not regularly updated .
these databases analyze pairwise interactions between chains or between domains as defined by scop ( 11 ) , cath ( 12 ) or pfam ( 13 ) .
those databases based on scop and cath are likely to be as behind on the pdb as scop and cath are , on the order of 12 years .
the current version of scop ( 1.75 ) covers only 38  221 entries or about 56% of the pdb .
cath ( v. 3.3 ) currently contains 53  132 entries from the pdb or 79% .
those databases that use only pdb , pqs and pisa provide search tools but usually no additional information on which interfaces in x - ray crystallographic structures are likely to be biologically relevant .
several other servers , such as prebi ( 14 ) , noxclass ( 15 ) , ibis ( 16 ) , pita ( 17 ) and dimovo ( 18 ) also analyze interfaces in pdb entries , and try to predict which interfaces may be biologically relevant . unlike pisa and pins , they characterize individual interfaces and do not try to predict assemblies larger than dimers .
an important feature of such servers is whether they are able to examine interfaces between monomers in different copies of the asymmetric unit and/or in different unit cells , since biologically relevant interactions may not be in the asymmetric unit itself ( 19 ) .
for instance , prebi identifies biological interfaces by analyzing the electrostatic potential , hydrophobicity , the shape and the area of the interfaces , including those between asymmetric units and 26 neighboring unit cells ( 14 ) .
the noxclass server uses support vector machines with features such as surface area , amino acid composition and conservation scores to infer biological relevance of interfaces , but only those within the asymmetric units of pdb entries ( 15 ) . the inferred biomolecular interaction server ( ibis )
predicts biologically relevant interactions for the chains in individual pdb entries by examining interfaces in the asymmetric units and the biological assemblies [ as annotated in pisa or the pdb or the conserved domain database ( 20 ) ] of proteins closely related to the query ( 16 ) . even if the shared interface is present in the query crystal , it is not annotated as 
pita works on pdb entries and uploaded pdb files and uses symmetry operators to find potential biological interfaces within crystals ( 17 ) .
however , it failed to find interfaces in structures with known oligomeric assemblies that we tried .
dimovo operates on uploaded pdb files containing dimers and uses solvation and voronoi tessellation to predict whether the interface is biological or not ( 18 ) .
it does not build additional unit cells and interfaces have to be uploaded one by one by the user .
we have previously shown that if an interface is present in a number of crystal forms ( cfs ) , especially when the proteins are homologous and not identical , then such interfaces are very likely to be biologically relevant structures ( 21 ) .
the data we analyzed previously were restricted to single - domain proteins separated into families as defined by scop and homologues of these identified with psi - blast . in this article
, we exploit this method for providing evidence in favor of the biological relevance of an interface across distant evolutionary relationships , and provide access to aligned structures of homo- and heterodimeric interfaces observed in multiple cfs .
the term architecture here is used in the same sense in which the pfam website uses it to denote the ordered pfam domain assignments along a protein sequence .
entry architecture that comprises the chain architectures in that entry . for each chain architecture observed in the pdb , we find all of the pdb entries and cfs with that chain architecture , and compare all of the homodimeric interfaces in representative entries of the different cfs .
we cluster these interfaces based on similarity of pairwise amino acid contacts using average linkage hierarchical clustering .
we perform the same kind of analysis on pairs of different chain architectures , comparing and clustering heterodimeric interfaces of single- and multi - domain protein chains .
a query to protcid is either a pdb entry code or a protein sequence or sequences .
the server returns the chain architecture(s ) for the query and asks the user to select one or two of these to search the database .
alternatively a user can browse a list of pfam families , which will identify all pdb entries with a particular pfam and their chain and entry architectures .
if a single chain architecture is chosen from the query , then the server returns a list of clusters of homodimeric interfaces ( and heterodimeric interfaces , if two chains have different sequences but both have the same chain architecture as the query ) .
if the query is a pair of chain architectures , the server returns a list of clusters of heterodimeric interfaces . in both cases
, for each cluster the server reports the number of cfs and pdb entries that contain the interface , the number of entries for which the pdb and pisa biological assemblies contain the interface , the average surface area and the minimum sequence identity between homologous chains in the different pdb entries in the cluster .
an expandable table for each cluster provides the list of pdb entries , and for each entry the cf , whether the pdb and pisa biological unit contains the interface , the interface surface area , and the name and species of the protein(s ) are provided .
first , it may be used to provide evidence in favor of the biological relevance of specific interfaces .
such interfaces are often conserved in different members of a family and may occur in different cfs .
rather , it provides data on common interfaces ( number of cfs , sequence information , pdb and pisa annotations , surface areas , etc . ) that may be used to complement biophysical analysis and experimental data . in benchmarking
, we found earlier that the more cfs that an interface is observed in , especially for non - identical proteins ( < 90% ) and interfaces over 400  , the more likely an interface was to be part of a biologically relevant assembly .
second , a user may be interested in using a particular pdb entry as a template for comparative modeling or for analysis of existing experimental data .
it commonly occurs that an interface shared by many pdb entries in a family is annotated in most but not all of the biological assemblies in the pdb and pisa .
it may still be present in the crystals of entries that are missing the annotation .
if this is the pdb entry of interest to a user , protcid may be used to identify the error and to provide coordinates for the interface from that entry .
it may sometimes occur that a protein  protein interaction is not stable under the crystallization conditions used for a particular pdb entry , and a common interface observed in family members related to the query may be of interest .
third , one of of protcid s most useful features is that the user can download coordinate files for each cluster with a single click .
we provide a program to produce a pymol script for each cluster to load the files , display them as rainbow - colored cartoon representations , and to align them to a common dimer in the cluster .
the resulting visual display is highly informative of the variation in the common interfaces within the protein family , and can be used as a basis for further study of the evolution of biologically important protein  protein interactions .
protcid is compiled from entries in the pdb using the pdbml ( xml ) formatted files ( 22 ) . from these files we obtain
coordinates , crystallographic symmetry , and the biological assemblies defined by the authors of each structure ( if available ) in terms of symmetry operators and asym_ids . since scop and cath are 12 years behind on the pdb , we annotate each sequence in the pdb with its pfam domains .
we obtain pfam domain assignments to sequences in the pdb from the pfam website ( 13 ) .
because the pfam website does not update its assignments to the pdb frequently , we use pfam s restful web service to assign pfams to entries missing from pfam itself .
we use the data from our pisces database ( 23,24 ) as well as the structure alignment program fatcat ( 23 ) .
pisces contains psi - blast ( 24 ) sequence alignments for all pdb chain pairs with e - values better than 0.001 [ based on profiles constructed from ncbi s non - redundant sequence database ( 25 ) ] and structure alignments using combinatorial extension ( ce ) ( 26 ) for those psi - blast detected homologues with sequence identity <
fatcat allows flexibility between protein domains so that two homologous multi - domain proteins may be aligned , even if the domains have somewhat different orientations in the two structures .
if both ce and fatcat alignments are available , we choose the alignment with higher score calculated by summing the blosum62 ( 27 ) substitution matrix scores over the aligned pairs .
biological units from the pdb are generated from information given in the pdb xml files , while pisa assemblies are generated from the xml descriptions from the pisa website .
the pdb s xml files contain biological assemblies from the authors and in many cases from pisa and pqs . at our suggestion
, the pdb now indicates the source of its biological assemblieswhether these are from the authors or from pisa or pqs . in some cases ,
the pisa website does not have a predicted assembly . although the pdb s xml file does contain a prediction from pisa ( from running the software locally at the pdb ) . in those cases ,
about 4% of pdb structures do not contain an author - approved biological assembly , and in these cases we use the software - generated assembly given in the pdb xml file that is from pisa or pqs .
for each pdb entry , there are two levels of pfam architectures : chain architecture and entry architecture .
chain architecture denotes the pfam domains in a protein chain with each pfam in parentheses and multiple pfams concatenated by_ in the order of their starting locations .
for instance , pdb entry 1e9h contains the sequence of cyclin a3 , which has the chain architecture 
( cyclin_n)_(cyclin_c). chains in a single entry with the same protein sequence ( and same entity_id in the pdb xml file ) are only represented once .
entry architecture is composed of the pfam architectures of all unique sequences in a pdb entry , sorted in alphabetical order and separated by semi - colons .
( cyclin_n)_(cyclin_c);(pkinase). to analyze homo- and heterodimeric interfaces , we create groups of pdb entries that contain particular chain architectures or pairs of chain architectures .
first , we define a group for each unique chain architecture found in one or more pdb entries .
these groups are used for analyzing homodimeric interfaces and some heterodimeric interfaces , those where both sequences are of the same chain architecture .
some of these entries have other proteins as well ( such as pkinase proteins ) but they all share the chain architecture  ( cyclin_n)_(cyclin_c). second , there is a group for each pair of chain architectures that occur together in at least one pdb entry .
so there is a group  ( cyclin_n)_(cyclin_c);(pkinase) containing 73 pdb entries that have these two proteins we divide each group into cfs , which are subsets of entries in the group with the same entry architectures , space group , the same asymmetric unit and similar crystal cell dimensions and angles ( 1% ) .
additionally we merged some cfs with the same entry architecture but different space groups or crystal cell dimensions or angles ( > 1% ) , if they contained highly similar interfaces , using a procedure described in our earlier work ( 21 ) .
we did not merge any cfs if the proteins had sequence identity less than 70% .
we used the entry in each cf with the best x - ray resolution as the representative entry of the cf to compare interfaces between cfs .
we build crystals from the asymmetric unit and space group defined in pdb xml files as described earlier ( 21 ) .
protein interactions are identified and analyzed from the crystals , and defined on the level of chains .
two chains are considered to be interacting if and only if they have at least 10 pairs of c atoms ( c for gly ) with distance  12 and at least one atomic contact 5 .
a  common interface indicates that a similar chain  chain interaction pattern occurs in at least two cfs .
the similarity of interface pairs was calculated by the q function described by xu et al .
( 21 ) , which is equal to a weighted count of the common contacting residue pairs in two interfaces divided by the total number of unique pairs . a value of q of 1 means all contacts in one interface exist in the other and at identical distances and vice versa .
a value of q of 0 means there are no common contacts . we have found that a value of q of 0.2 or higher usually indicates a common interface and common orientations of the two monomers .
we cluster interfaces of representative entries with surface area > 200  using a hierarchical average linkage clustering algorithm ( 28 ) . in this method ,
each interface is initialized to be a cluster . at each step , the two clusters with the highest average q - score are merged , as long as , where   is the average q - score between two clusters : 

there are a total of 6067 pfam ( v24.0 ) families represented in protein sequences in the pdb .
these exist in single and multi - domain proteins to form 7463 different chain architectures with known structure .
groups of pdb entries based on individual chain architectures or pairs of chain architectures to investigate interfaces in homo- and heterodimers , respectively . in this way
, there are 7463 groups with a single architecture and 5768 pair architectures for a total of 13  231 pfam architecture groups .
there are a total of 3224 pfam architecture groups with common interfaces (  clusters ) in more than one cf , comprising 39  871 distinct pdb entries and 11  402 clusters .
a total of 11% of these ( 1223 clusters ) contain heterodimeric interfaces showing the interactions between different pfam architectures .
the number of cfs in a group ranges from 1 to 591 [ the antibody group ( v - set)_(c1-set ) ] and 50% of groups have two or three cfs ( figure 1 top ) . in this study
, we do not analyze the groups that contain only v - set and c1-set .
a total of 133 groups contain at least one common interface that exists in all of 10 or more distinct cfs ( figure 1 bottom ) . 

top , the number of groups given for each of the number of cfs in that group ( only when # cf>1 ) .
bottom , the number of groups that contain interfaces present in all n out of n cfs for that group . 
 table 1.summary of data in protcidsingle chain architecturespairs of chain architecturestotal # groups7463576813  231 # groups with m    224588133271 # groups with m    2 , seqid<9014613171778 # groups with m    5 , seqid<90615116731 # groups with m    10 , seqid<9025545300 # groups with m    20 , seqid<9088492 # entries62  499598262  499 # entries with m    239  076427040  216 # entries with m    2 , seqid<9029  282298430  382 # entries with m    5 , seqid<9019  935216620  877 # entries with m    10 , seqid<9013  301138014  294 # entries with m    20 , seqid<9077547328408 # clusters with m    210  761130812  069 # clusters with m    2,seqid<9056965116207 # clusters with m    5,seqid<9014031611195 # clusters with m    10,seqid<9035555410 # clusters with m   
top , the number of groups given for each of the number of cfs in that group ( only when # cf>1 ) .
bottom , the number of groups that contain interfaces present in all n out of n cfs for that group . summary of data in protcid m is the number of cfs that contain a common interface .
there are three types of queries : ( i ) a pdb i d ; ( ii ) a pfam i d or accession code ; and ( iii ) one or two protein sequences .
a pdb i d query , such as 1m54 as shown in figure 2 ( top ) , a crystal structure of cystathionine -synthase ( 29 ) , will return a list of pfam chain architectures present in that pdb , in this case simply 
( palp) for pyridoxal - phosphate dependent enzymes ( figure 2 , just below the first arrow ) .
a user can select this pfam chain architecture using the checkbox , and the server will return the interface clusters and the details about each cluster for the architecture ( figure 2 , just below the second arrow ) .
the first cluster of interfaces occurs in 37 cfs , or 71% of 52 cfs for the chain architecture ( palp ) , and 109 pdb entries .
this interface occurs in the asymmetric units of only 42 of these 109 entries ( 39% ) , demonstrating the need for investigating interfaces induced by symmetry relationships in a crystal . clicking on the cluster number in the second column
protcid provides a perl script for creating a structure superposition in pymol ( delano , w.l .
, http://pymol.org ) from the files in a folder containing the pdb files with the dimer interfaces .
this dimer is well annotated with the interface observed in 103 and 105 of the pdb and pisa biological assemblies , respectively .
notably a search of the ibis site at ncbi using 1m54 as a query provides the same dimer interface shown in figure 3a but deems the entry a 
singleton and does not show any of the interfaces in the 25 different but related proteins shown in figure 3a .
the reason for this is that ibis has a relatively conservative cutoff for sequence identity with respect to the query of 50% for homodimers and 30% for heterodimers .
ibis does show this dimer for almost all of the other entries in this family .
pisa can be used to find similar interfaces in homologues in the pdb but a search with 1m54 took several hours to complete , and pisa does not provide information on cfs . 

the search returns the pfam architecture of 1m54 , ( palp ) , or pyridoxal - dependent enzymes . clicking the box for this pfam architecture and clicking the button labeled 
retrieve common interface clusters produces a table of clusters in the lower part of the figure . clicking on  +  next to a cluster produces a table of the pdb entries that contain the common interface in that cluster . 

( a ) a cluster of interfaces present in 37 cfs and 109 pdb entries ( only one structure per cf is shown ) .
these structures include cysteine synthase , tryptophan synthase alpha subunit , threonine synthase , cystathionine beta synthase , 1-aminocyclopropane-1-carboxylate deaminase and o - acetylserine sulfhydrylase from 18 different species .
( b ) a cluster of interfaces present in 13 cfs and 17 pdb entries .
these proteins include catabolic threonine dehydratase , serine racemase , threonine deaminase , l - serine dehydratase and two uncharacterized proteins from schizosaccharomyces pombe .
. the search returns the pfam architecture of 1m54 , ( palp ) , or pyridoxal - dependent enzymes . clicking the box for this pfam architecture and clicking the button labeled 
retrieve common interface clusters produces a table of clusters in the lower part of the figure . clicking on  +  next to a cluster produces a table of the pdb entries that contain the common interface in that cluster .
( a ) a cluster of interfaces present in 37 cfs and 109 pdb entries ( only one structure per cf is shown ) .
these structures include cysteine synthase , tryptophan synthase alpha subunit , threonine synthase , cystathionine beta synthase , 1-aminocyclopropane-1-carboxylate deaminase and o - acetylserine sulfhydrylase from 18 different species .
( b ) a cluster of interfaces present in 13 cfs and 17 pdb entries .
these proteins include catabolic threonine dehydratase , serine racemase , threonine deaminase , l - serine dehydratase and two uncharacterized proteins from schizosaccharomyces pombe .
a second large cluster for ( palp ) is listed in the screenshot of protcid in figure 2 , which exists in 13 cfs and 17 pdb entries .
these structures do not overlap with the set in cluster 1 for ( palp ) and form a different branch on a phylogenetic tree as shown in figure 4 .
the pdb and pisa have 11 and 13 of these interfaces in their biological assemblies for these entries , respectively .
both miss this interface for the human and rat l - serine dehydratases in three different cfs .
an ibis search with pdb entry 3hmk in this cluster provides 5 of the 15 pdb entries ( and 3 of 7 genes ) shown by protcid . 

figure 4.a phylogenetic tree of the proteins in the pdb with the pfam architecture ( palp ) .
only two threonine synthase structures , pdb entries 1vb3 ( escherichia coli ) and 1kl7 ( yeast ) , are missing either common dimer in their x - ray structures .
the numbers after the pdb codes indicate the pdb s entity_id number . a phylogenetic tree of the proteins in the pdb with the pfam architecture ( palp ) .
only two threonine synthase structures , pdb entries 1vb3 ( escherichia coli ) and 1kl7 ( yeast ) , are missing either common dimer in their x - ray structures .
we previously analyzed single - domain proteins as defined by scop ( 11 ) , but these results were limited to that portion of the pdb covered by scop and proteins related to scop - defined domains as calculated by psi - blast ( 21 ) . by using pfam to identify multi - domain proteins , instead of scop , we are able to classify the domains in 98% of pdb entries and 98% of sequences in the pdb . in order to achieve this coverage
, we used pfam s restful web service ( 13 ) , since pfam itself is several months behind on the pdb .
while in general scop is able to identify more remote relationships than pfam , we are interested in common interfaces related by evolution . at longer evolutionary distances ,
some interfaces are not conserved ( 30 ) and so we do not necessarily require the identification of the most remote relationships . of the 83  539 ( redundant ) entity sequences in the pdb ,
protcid can be searched ( or browsed ) by pfam families and thus multi - domain proteins that share a particular domain can be readily identified .
pdz is entered , a list of 293 pdb entries is returned along with their pfam chain and entry architectures .
there are nine different chain architectures in the pdb that contain a pdz domain : ( pdz ) , 240 entries ; ( trypsin)_(pdz ) , 14 entries ; ( pdz)_(pdz ) , 11 entries ; ( pdz_assoc)_(pdz ) , 8 entries ; ( trypsin)_(pdz)_(pdz ) , 6 entries ; ( maguk_n_pest)_(pdz ) , 6 entries ; ( pdz)_(peptidase_s41 ) , 5 entries ; ( pdz)_(fn3 ) , 2 entries ; and ( pdz)_(ebp50_c - term ) , 1 entry . if we select one of the entries that has a chain architecture ( trypsin)_(pdz ) , we find a cluster of homodimeric interfaces that occurs in all seven cfs and 14 entries for this architecture .
protcid shows that these proteins are annotated mostly as trimers in the pdb and pisa , and the interfaces in the figure are in fact the trimer interface . in the pdb ,
one of these , the structure of human htra2 serine protease , a mitochondrial protein involved in apoptosis ( 31 ) , is described as a trimer in the published paper ( 32 ) , while the biological assembly in the pdb ( pdb entry 1lcy ) was deposited as a monomer .
this is an example where the author - deposited biological assemblies in the pdb do not necessarily coincide with what the authors themselves may view as the biologically relevant structure .
the reasons for this are not clear but the phenomenon is not uncommon in our experience .
protcid is valuable in checking whether a pdb entry of interest contains a biologically relevant interface present in many cfs of related proteins but for some reason is missing from the publicly available annotations . in this case
the structure of the htra2 trimer is available from pisa or can be built in pymol or other programs from the crystal symmetry operators .
ibis does not have this interaction for pdb entry 1lcy because of its conservative sequence identity cutoff . 

figure 5.homodimers of a multi - domain protein with pfam architecture ( trypsin)_(pdz ) from seven cfs and 14 pdb entries .
the proteins include e. coli proteases degs and do , mycobacterium tuberculosis hypothetical protein rv0983 and human htra2 .
. homodimers of a multi - domain protein with pfam architecture ( trypsin)_(pdz ) from seven cfs and 14 pdb entries .
the proteins include e. coli proteases degs and do , mycobacterium tuberculosis hypothetical protein rv0983 and human htra2 .
the pdz domains are in orange and red . in some cases , pisa underannotates well - documented biologically relevant interfaces . in the case of another multi - domain protein , with pfam chain architecture ( atp - gua_ptransn)_(atp - gua_ptrans ) ,
a single interface is present in 14 of 18 cfs and 18 pdb entries with minimum sequence identity of 49% .
of these entries , the pdb s biological units contain this dimer in 14 of 18 entries , while pisa has this dimer in only five cases .
three of the incorrect dimers in the pdb are in a single cf of human creatine kinase b. in their paper ( 33 ) , the authors describe the common dimer shown by protcid , but the deposited biological assembly for all three ( pdb entries 3b6r , 3drb and 3dre ) is the same as the asymmetric unit for this cf , another example of apparently accidental misannotations in the pdb .
biologically validated for four pdb entries ( 3ju5 , 3ju6 , 3l2d , 1u6r ) and as  putative but not biologically validated in five more ( 2gl6 , 3l2f , 1qh4 1qk1 , 1vrp ) . 

figure 6.homodimers of pfam chain architecture ( atp_gua_ptransn)_(atp_gua_ptrans ) from 14 cfs and 18 pdb entries .
the proteins include human , rabbit , chicken , bovine and electric ray creatine kinase , a worm glycocyamine kinase and a sea cucumber arginine kinase .
homodimers of pfam chain architecture ( atp_gua_ptransn)_(atp_gua_ptrans ) from 14 cfs and 18 pdb entries .
the proteins include human , rabbit , chicken , bovine and electric ray creatine kinase , a worm glycocyamine kinase and a sea cucumber arginine kinase .
a search starting with pdb entry 2gtp ( 34 ) reveals that this entry has two sequences , one with pfam architecture , ( rgs ) , the regulator of g - protein signaling , and one with pfam ( g - alpha ) , the g - protein -subunit .
the first of these is present in eight pdb entries and all six cfs with a minimum sequence identity of 36% .
the second cluster is in only two cfs with 100% sequence identity and is not likely to be biologically relevant .
we found previously that identical proteins may form the same interface in different cfs , although this is rare for homologous but not identical proteins .
this dimer is annotated in the deposited biological units of all eight pdb entries in these cfs but only one of the pisa assemblies contains this interface .
six of the entries missing the heterodimer in pisa are oddly monomeric , and not heterooligomers at all .
one is a heterodimer when it should be a heterotrimer ( pdb entry 1fqj ) . 

figure 7.heterodimers of pfam domains ( g - alpha ) , the g - protein alpha subunit , and ( rgs ) , the regulator of g - protein signaling , from six cfs and eight pdb entries .
the rgs proteins include human rgs1 , rgs2 , rgs8 , rgs9 , rgs10 and rgs16 as well as bovine rgs9 and mouse rgs16 .
the g - protein alpha subunits include human gi and gk , bovine gt and mouse go .
heterodimers of pfam domains ( g - alpha ) , the g - protein alpha subunit , and ( rgs ) , the regulator of g - protein signaling , from six cfs and eight pdb entries .
the rgs proteins include human rgs1 , rgs2 , rgs8 , rgs9 , rgs10 and rgs16 as well as bovine rgs9 and mouse rgs16 .
the g - protein alpha subunits include human gi and gk , bovine gt and mouse go .
ibis shows this same dimer in all of the eight pdb entries that contain it .
ibis also has results for 2gtp that include many of the interactions for ( g - alpha ) proteins in protcid , including interactions with ( rgs_like ) , ( rhogef ) , ( guanylate_cyc ) and ( pde6_gamma ) .
ibis presents some results for the query 2gtp that protcid does not due to the way pfam divides the aaa superfamily of proteins . in pfam , ( g - alpha ) and
( arf ) are two families in the aaa clan . since we do not yet compare chains with different pfams even when they are in the same clan , ( arf)-containing proteins are not included in the results for any g - alpha entries .
ibis does include some arf proteins in the results for 2gtp , including interaction with pleckstrin homology domains .
ibis also provides an intraprotein interaction between an arf domain and an arfgap domain in pdb entry 3lvq , which protcid does not provide , since we do not currently analyze intraprotein domain  domain interfaces .
there are a total of 6067 pfam ( v24.0 ) families represented in protein sequences in the pdb .
these exist in single and multi - domain proteins to form 7463 different chain architectures with known structure .
groups of pdb entries based on individual chain architectures or pairs of chain architectures to investigate interfaces in homo- and heterodimers , respectively . in this way
, there are 7463 groups with a single architecture and 5768 pair architectures for a total of 13  231 pfam architecture groups .
there are a total of 3224 pfam architecture groups with common interfaces (  clusters ) in more than one cf , comprising 39  871 distinct pdb entries and 11  402 clusters .
a total of 11% of these ( 1223 clusters ) contain heterodimeric interfaces showing the interactions between different pfam architectures .
the number of cfs in a group ranges from 1 to 591 [ the antibody group ( v - set)_(c1-set ) ] and 50% of groups have two or three cfs ( figure 1 top ) . in this study
, we do not analyze the groups that contain only v - set and c1-set .
a total of 133 groups contain at least one common interface that exists in all of 10 or more distinct cfs ( figure 1 bottom ) . 

top , the number of groups given for each of the number of cfs in that group ( only when # cf>1 ) .
bottom , the number of groups that contain interfaces present in all n out of n cfs for that group . 
 table 1.summary of data in protcidsingle chain architecturespairs of chain architecturestotal # groups7463576813  231 # groups with m    224588133271 # groups with m    2 , seqid<9014613171778 # groups with m    5 , seqid<90615116731 # groups with m    10 , seqid<9025545300 # groups with m    20 , seqid<9088492 # entries62  499598262  499 # entries with m    239  076427040  216 # entries with m    2 , seqid<9029  282298430  382 # entries with m    5 , seqid<9019  935216620  877 # entries with m    10 , seqid<9013  301138014  294 # entries with m    20 , seqid<9077547328408 # clusters with m    210  761130812  069 # clusters with m    2,seqid<9056965116207 # clusters with m    5,seqid<9014031611195 # clusters with m    10,seqid<9035555410 # clusters with m   
top , the number of groups given for each of the number of cfs in that group ( only when # cf>1 ) .
bottom , the number of groups that contain interfaces present in all n out of n cfs for that group . summary of data in protcid m is the number of cfs that contain a common interface .
there are three types of queries : ( i ) a pdb i d ; ( ii ) a pfam i d or accession code ; and ( iii ) one or two protein sequences .
a pdb i d query , such as 1m54 as shown in figure 2 ( top ) , a crystal structure of cystathionine -synthase ( 29 ) , will return a list of pfam chain architectures present in that pdb , in this case simply 
( palp) for pyridoxal - phosphate dependent enzymes ( figure 2 , just below the first arrow ) .
a user can select this pfam chain architecture using the checkbox , and the server will return the interface clusters and the details about each cluster for the architecture ( figure 2 , just below the second arrow ) .
the first cluster of interfaces occurs in 37 cfs , or 71% of 52 cfs for the chain architecture ( palp ) , and 109 pdb entries .
this interface occurs in the asymmetric units of only 42 of these 109 entries ( 39% ) , demonstrating the need for investigating interfaces induced by symmetry relationships in a crystal .
clicking on the cluster number in the second column downloads a gzipped tar file with the coordinates of every dimer in the cluster .
protcid provides a perl script for creating a structure superposition in pymol ( delano , w.l .
, http://pymol.org ) from the files in a folder containing the pdb files with the dimer interfaces .
this dimer is well annotated with the interface observed in 103 and 105 of the pdb and pisa biological assemblies , respectively .
notably a search of the ibis site at ncbi using 1m54 as a query provides the same dimer interface shown in figure 3a but deems the entry a 
singleton and does not show any of the interfaces in the 25 different but related proteins shown in figure 3a .
the reason for this is that ibis has a relatively conservative cutoff for sequence identity with respect to the query of 50% for homodimers and
ibis does show this dimer for almost all of the other entries in this family .
pisa can be used to find similar interfaces in homologues in the pdb but a search with 1m54 took several hours to complete , and pisa does not provide information on cfs . 

the search returns the pfam architecture of 1m54 , ( palp ) , or pyridoxal - dependent enzymes . clicking the box for this pfam architecture and clicking the button labeled 
retrieve common interface clusters produces a table of clusters in the lower part of the figure .
clicking on  +  next to a cluster produces a table of the pdb entries that contain the common interface in that cluster . 

( a ) a cluster of interfaces present in 37 cfs and 109 pdb entries ( only one structure per cf is shown ) .
these structures include cysteine synthase , tryptophan synthase alpha subunit , threonine synthase , cystathionine beta synthase , 1-aminocyclopropane-1-carboxylate deaminase and o - acetylserine sulfhydrylase from 18 different species .
( b ) a cluster of interfaces present in 13 cfs and 17 pdb entries .
these proteins include catabolic threonine dehydratase , serine racemase , threonine deaminase , l - serine dehydratase and two uncharacterized proteins from schizosaccharomyces pombe .
the search returns the pfam architecture of 1m54 , ( palp ) , or pyridoxal - dependent enzymes . clicking the box for this pfam architecture and clicking the button labeled 
retrieve common interface clusters produces a table of clusters in the lower part of the figure . clicking on  +  next to a cluster produces a table of the pdb entries that contain the common interface in that cluster .
( a ) a cluster of interfaces present in 37 cfs and 109 pdb entries ( only one structure per cf is shown ) .
these structures include cysteine synthase , tryptophan synthase alpha subunit , threonine synthase , cystathionine beta synthase , 1-aminocyclopropane-1-carboxylate deaminase and o - acetylserine sulfhydrylase from 18 different species .
( b ) a cluster of interfaces present in 13 cfs and 17 pdb entries .
these proteins include catabolic threonine dehydratase , serine racemase , threonine deaminase , l - serine dehydratase and two uncharacterized proteins from schizosaccharomyces pombe .
a second large cluster for ( palp ) is listed in the screenshot of protcid in figure 2 , which exists in 13 cfs and 17 pdb entries .
these structures do not overlap with the set in cluster 1 for ( palp ) and form a different branch on a phylogenetic tree as shown in figure 4 .
the pdb and pisa have 11 and 13 of these interfaces in their biological assemblies for these entries , respectively .
both miss this interface for the human and rat l - serine dehydratases in three different cfs .
an ibis search with pdb entry 3hmk in this cluster provides 5 of the 15 pdb entries ( and 3 of 7 genes ) shown by protcid . 

figure 4.a phylogenetic tree of the proteins in the pdb with the pfam architecture ( palp ) .
only two threonine synthase structures , pdb entries 1vb3 ( escherichia coli ) and 1kl7 ( yeast ) , are missing either common dimer in their x - ray structures .
the numbers after the pdb codes indicate the pdb s entity_id number . a phylogenetic tree of the proteins in the pdb with the pfam architecture ( palp ) .
only two threonine synthase structures , pdb entries 1vb3 ( escherichia coli ) and 1kl7 ( yeast ) , are missing either common dimer in their x - ray structures .
we previously analyzed single - domain proteins as defined by scop ( 11 ) , but these results were limited to that portion of the pdb covered by scop and proteins related to scop - defined domains as calculated by psi - blast ( 21 ) . by using pfam to identify multi - domain proteins , instead of scop , we are able to classify the domains in 98% of pdb entries and 98% of sequences in the pdb . in order to achieve this coverage
, we used pfam s restful web service ( 13 ) , since pfam itself is several months behind on the pdb .
while in general scop is able to identify more remote relationships than pfam , we are interested in common interfaces related by evolution . at longer evolutionary distances ,
some interfaces are not conserved ( 30 ) and so we do not necessarily require the identification of the most remote relationships . of the 83  539 ( redundant )
protcid can be searched ( or browsed ) by pfam families and thus multi - domain proteins that share a particular domain can be readily identified .
pdz is entered , a list of 293 pdb entries is returned along with their pfam chain and entry architectures .
there are nine different chain architectures in the pdb that contain a pdz domain : ( pdz ) , 240 entries ; ( trypsin)_(pdz ) , 14 entries ; ( pdz)_(pdz ) , 11 entries ; ( pdz_assoc)_(pdz ) , 8 entries ; ( trypsin)_(pdz)_(pdz ) , 6 entries ; ( maguk_n_pest)_(pdz ) , 6 entries ; ( pdz)_(peptidase_s41 ) , 5 entries ; ( pdz)_(fn3 ) , 2 entries ; and ( pdz)_(ebp50_c - term ) , 1 entry .
if we select one of the entries that has a chain architecture ( trypsin)_(pdz ) , we find a cluster of homodimeric interfaces that occurs in all seven cfs and 14 entries for this architecture .
protcid shows that these proteins are annotated mostly as trimers in the pdb and pisa , and the interfaces in the figure are in fact the trimer interface . in the pdb ,
one of these , the structure of human htra2 serine protease , a mitochondrial protein involved in apoptosis ( 31 ) , is described as a trimer in the published paper ( 32 ) , while the biological assembly in the pdb ( pdb entry 1lcy ) was deposited as a monomer .
this is an example where the author - deposited biological assemblies in the pdb do not necessarily coincide with what the authors themselves may view as the biologically relevant structure .
the reasons for this are not clear but the phenomenon is not uncommon in our experience .
protcid is valuable in checking whether a pdb entry of interest contains a biologically relevant interface present in many cfs of related proteins but for some reason is missing from the publicly available annotations . in this case
the structure of the htra2 trimer is available from pisa or can be built in pymol or other programs from the crystal symmetry operators .
ibis does not have this interaction for pdb entry 1lcy because of its conservative sequence identity cutoff . 

figure 5.homodimers of a multi - domain protein with pfam architecture ( trypsin)_(pdz ) from seven cfs and 14 pdb entries .
the proteins include e. coli proteases degs and do , mycobacterium tuberculosis hypothetical protein rv0983 and human htra2 .
homodimers of a multi - domain protein with pfam architecture ( trypsin)_(pdz ) from seven cfs and 14 pdb entries .
the proteins include e. coli proteases degs and do , mycobacterium tuberculosis hypothetical protein rv0983 and human htra2 .
the pdz domains are in orange and red . in some cases , pisa underannotates well - documented biologically relevant interfaces . in the case of another multi - domain protein , with pfam chain architecture ( atp - gua_ptransn)_(atp - gua_ptrans ) ,
a single interface is present in 14 of 18 cfs and 18 pdb entries with minimum sequence identity of 49% .
of these entries , the pdb s biological units contain this dimer in 14 of 18 entries , while pisa has this dimer in only five cases .
three of the incorrect dimers in the pdb are in a single cf of human creatine kinase b. in their paper ( 33 ) , the authors describe the common dimer shown by protcid , but the deposited biological assembly for all three ( pdb entries 3b6r , 3drb and 3dre ) is the same as the asymmetric unit for this cf , another example of apparently accidental misannotations in the pdb .
biologically validated for four pdb entries ( 3ju5 , 3ju6 , 3l2d , 1u6r ) and as  putative but not biologically validated in five more ( 2gl6 , 3l2f , 1qh4 1qk1 , 1vrp ) . 

figure 6.homodimers of pfam chain architecture ( atp_gua_ptransn)_(atp_gua_ptrans ) from 14 cfs and 18 pdb entries .
the proteins include human , rabbit , chicken , bovine and electric ray creatine kinase , a worm glycocyamine kinase and a sea cucumber arginine kinase .
homodimers of pfam chain architecture ( atp_gua_ptransn)_(atp_gua_ptrans ) from 14 cfs and 18 pdb entries .
the proteins include human , rabbit , chicken , bovine and electric ray creatine kinase , a worm glycocyamine kinase and a sea cucumber arginine kinase .
a search starting with pdb entry 2gtp ( 34 ) reveals that this entry has two sequences , one with pfam architecture , ( rgs ) , the regulator of g - protein signaling , and one with pfam ( g - alpha ) , the g - protein -subunit .
the first of these is present in eight pdb entries and all six cfs with a minimum sequence identity of 36% .
the second cluster is in only two cfs with 100% sequence identity and is not likely to be biologically relevant .
we found previously that identical proteins may form the same interface in different cfs , although this is rare for homologous but not identical proteins .
this dimer is annotated in the deposited biological units of all eight pdb entries in these cfs but only one of the pisa assemblies contains this interface .
six of the entries missing the heterodimer in pisa are oddly monomeric , and not heterooligomers at all .
one is a heterodimer when it should be a heterotrimer ( pdb entry 1fqj ) . 

figure 7.heterodimers of pfam domains ( g - alpha ) , the g - protein alpha subunit , and ( rgs ) , the regulator of g - protein signaling , from six cfs and eight pdb entries .
the rgs proteins include human rgs1 , rgs2 , rgs8 , rgs9 , rgs10 and rgs16 as well as bovine rgs9 and mouse rgs16 .
the g - protein alpha subunits include human gi and gk , bovine gt and mouse go .
heterodimers of pfam domains ( g - alpha ) , the g - protein alpha subunit , and ( rgs ) , the regulator of g - protein signaling , from six cfs and eight pdb entries .
the rgs proteins include human rgs1 , rgs2 , rgs8 , rgs9 , rgs10 and rgs16 as well as bovine rgs9 and mouse rgs16 .
the g - protein alpha subunits include human gi and gk , bovine gt and mouse go .
ibis shows this same dimer in all of the eight pdb entries that contain it .
ibis also has results for 2gtp that include many of the interactions for ( g - alpha ) proteins in protcid , including interactions with ( rgs_like ) , ( rhogef ) , ( guanylate_cyc ) and ( pde6_gamma ) .
ibis presents some results for the query 2gtp that protcid does not due to the way pfam divides the aaa superfamily of proteins . in pfam , ( g - alpha ) and
( arf ) are two families in the aaa clan . since we do not yet compare chains with different pfams even when they are in the same clan , ( arf)-containing proteins are not included in the results for any g - alpha entries .
ibis does include some arf proteins in the results for 2gtp , including interaction with pleckstrin homology domains .
ibis also provides an intraprotein interaction between an arf domain and an arfgap domain in pdb entry 3lvq , which protcid does not provide , since we do not currently analyze intraprotein domain  domain interfaces .
some interactions are weak or transient and methods designed to identify stable protein  protein interfaces in crystals using physical chemical considerations may not identify these easily . in our earlier work
, we showed that these methods also tend to identify some very large surface areas in crystals as biologically relevant even when the proteins are monomeric by all available experimental data ( 21 ) .
we provide protcid as an alternative and complementary source of information on the biologically relevant interfaces between proteins in x - ray crystal structures .
when there is some doubt about an interface , observing the same interface in homologous proteins may provide evolutionary and physical evidence in favor of that interface . as more structures are determined in large protein families
, the comparison of cfs may play an important role in suggesting which interfaces are biologically relevant interactions . identifying such common interfaces is difficult to perform manually for many structures , and therefore protcid provides a useful tool .
we hope that the common interfaces identified by protcid can be used in further studies of the biophysical characteristics and evolutionary conservation of interaction surfaces for specific biological systems .
first , in cases where there is only one cf for a protein or protein complex , protcid will not have any common interfaces to report .
this occurs for 36% of the pdb , of which 32% are annotated as multimeric by pisa .
second , it sometimes occurs that similar interfaces are present in more than one cf but are not biologically relevant .
this usually occurs when the entries are for identical sequences , for small interfaces less than 400  , and usually for only two or three cfs .
it may also occur for families with large numbers of structures , where through thorough sampling some interfaces may show high similarity by chance .
third , any such server needs to define such relationships and strike a balance between clustering related proteins without introducing false relationships .
we have used pfam to define protein domains and architectures and therefore the evolutionary relationships among proteins .
. thus some distant evolutionary relationships may be missed in protcid . finally , we compared proteins with the same architectures ( either one or two at a time ) .
so , for instance , we would not compare homodimeric interfaces between entries with architecture ( pkinase_tyr)_(sh2 ) and those with ( pkinase_tyr ) .
the reason for this is that while pfam may identify most proteins that belong to its defined domain families , it often aligns regions shorter than the full domain as observed in the three - dimensional structure .
protcid will be updated monthly in order to keep up with the rapidly expanding size of the pdb .
a comparison of interactions on the domain level , instead of the chain level , will be presented later .
national institutes of health ( r01-gm73784 and r01-gm84453 to r.d . ) . funding for open access charge : national institutes of health ( r01-gm73784 ) .
 the protein common interface database ( protcid ) is a database that contains clusters of similar homodimeric and heterodimeric interfaces observed in multiple crystal forms ( cfs ) . 
 such interfaces , especially of homologous but non - identical proteins , have been associated with biologically relevant interactions . in protcid , 
 protein chains in the protein data bank ( pdb ) are grouped based on their pfam domain architectures . for a single pfam architecture , 
 all the dimers present in each cf are constructed and compared with those in other cfs that contain the same domain architecture . 
 interfaces occurring in two or more cfs comprise an interface cluster in the database . 
 the same process is used to compare heterodimers of chains with different domain architectures . by examining interfaces that are shared by many homologous proteins in different cfs , we find that the pdb and the protein interfaces , surfaces , and assemblies ( pisa ) are not always consistent in their annotations of biological assemblies in a homologous family . 
 our data therefore provide an independent check on publicly available annotations of the structures of biological interactions for pdb entries . 
 common interfaces may also be useful in studies of protein evolution . 
 coordinates for all interfaces in a cluster are downloadable for further analysis . 
 protcid is available at http://dunbrack2.fccc.edu/protcid . 
INTRODUCTION
METHODS
RESULTS
Summary statistics of the ProtCiD database
Searching ProtCID
DISCUSSION
FUNDING