BACKGROUND
the most common biologically-relevant mutations are single base changes often referred to as single nucleotide polymorphisms .
these account for about 90% of sequence polymorphisms in humans  <cit>  at an overall frequency of about one per  <dig> bases  <cit> .
traditionally, snps are classified as coding or non-coding according to their genomic location – coding snps are further sub-classified according to the protein product expressed.
non-synonymous snps  are those that alter the amino acid sequence of the protein product, either through amino acid substitution , or by the generation of truncation mutations.
by contrast, synonymous snps  are those that do not alter the amino acid sequence of the protein product.
not all synonymous snps are neutral since they may still affect the expression of gene products or protein translation by introducing alterations into regulatory regions, interfering with splice sites or impinging on other regulatory mechanisms  <cit> .
similarly, not all nssnps are associated with pathological diseases, since some changes are, by nature, milder than others, and diseases commonly involve complex sets of alterations.
strictly the term 'snp' is defined as a mutation which occurs in at least 1% of a 'normal' population.
thus snps are expected to have a neutral non-deleterious or low-penetrance phenotypic effect whereas the term pathogenic deviation  refers to those mutations that generally occur at much lower frequencies in the population and have a severe effect on phenotype.
the most commonly used database for storing information on snps is dbsnp  <cit> , which currently contains several million validated snps from humans and other species.
other sources of genomic information about snps include ensembl  <cit>  and the hapmap project  <cit> .
several efforts have been devoted to the prediction of the pathogenicity of amino acid mutations, resulting from single nucleotide changes.
these methods make use of a set of characteristics which may be based both on sequence and structure, to determine whether a mutation can affect protein function and therefore be, potentially, associated with disease.
this is an area of active research as shown by the considerable number of publications on the subject during the last few years  <cit> .
several efforts, saapdb  <cit>  among others, have been devoted to compiling this information and to providing a sequence and structural analysis, where possible, aiming to determine the origin of the pathogenicity shown.
in this type of repository, the term snp is used to refer to essentially phenotypically silent mutations, while pd is used for mutations known to have a severe effect on phenotype, i.e.
any single base change reported to correlate with disease.
online mendelian inheritance in man   <cit>  is a collection of information about inherited disease and contains data on pds.
however a great deal more information is held and maintained by individual research groups in locus-specific mutation databases or lsmdbs  <cit> .
like pds, nssnps are point mutations, but by definition they occur in at least 1% of a 'normal' population.
they are expected to have a neutral non-deleterious or low-pentrance phenotypic effect whereas pds are known to be detrimental.
by mapping these saaps  onto protein structures, we can begin to understand how protein structure might be affected by mutant residues, and so begin to explain the functional effect  of the mutation.
saapdb provides potential explanations for both pds, derived from various sources, and snps, derived from dbsnp  <cit> .
the cath  <cit>  structural domain database is a manually curated classification of domain structures found in the protein data bank   <cit> , grouped according to evolutionary relationships and structural features.
hidden markov models  are derived from alignments of these structural exemplars and used by gene3d  <cit>  to identify homologues within the protein sequences of uniprot  <cit> , refseq  <cit>  and ensembl  <cit> .
here we present 3dsim , a system mapping single amino-acid polymorphisms onto structures of cath domains.
for sequences with no known structure, the gene3d resource of domain structure annotations is used to map the sequence onto the closest homologous domain of known structure in cath.
thus 3dsim is of particular interest when no structural information is available for a protein in which mutations are known to occur as it uses sequence homology to map to the closest representative structure.
this provides a comprehensive overview of the distribution of mutations in structural space, as well as a visualization tool for pinpointing the locations of mutations on individual structures rendered in jmol , as well as links to detailed information on each sequence, structure and mutation.
the 3dsim application, which was designed with the aim of being very intuitive, easy to use and user-friendly, is publicly available at .
several worked examples are available, along with a 6-minute video tutorial.
in addition, for those advanced users needing intensive programmatic access to the information stored, the underlying database containing the mappings between saapdb, gene3d and cath is available on request, and most of the functionality is available as web services implemented in soap.
RESULTS
the mapping between saapdb and gene3d
saapdb contains polymorphism data for  <dig> sequences without a structure.
almost all of these could be mapped to gene3d:  <dig> identical sequences were found in the gene3d database.
of the remaining  <dig>   <dig> had sequences with the same length and associated uniprot accession, leaving only  <dig> for which a reliable match could not be obtained directly.
the mapping between gene3d and cath
where no structural data are available, the best representative cath domain is selected on the basis of homology.
for each of the  <dig> superfamilies in cath, a database of all cath domains was built.
for each of the  <dig> gene3d domain sequences mapped to cath structural superfamilies for which there is information about mutations in saapdb , a blast  <cit>  search was run against the corresponding superfamily database.
the closest relative found  was used to cluster the sequences.
sequences with a sequence identity less than 20% were placed in separate clusters.
this process yielded  <dig> different groups.
the groups  were then aligned using muscle  <cit>  and the resulting alignments used to transfer the mutations from gene3d sequences to cath domain representative structures.
at the end of the pipeline we were able to display information on  <dig> point mutations,  <dig> of them known to be associated with disease, mapping to  <dig> cath superfamilies.
the complete pipeline is described in figure  <dig> and details are provided in the methods section.
description of web application
the initial input for the system is a cath superfamily identifier for which the user wants to retrieve information on mapped mutations.
alternatively, the database can be searched using uniprot accession codes or cath domain identifiers.
the user can either manually introduce the desired superfamily identifier in the provided form, or browse the superfamilies in cath in order to access the information.
after this initial step, information about the selected cath superfamily is displayed along with the cath domains for which there is information about mutations in saapdb.
in addition, for users that are interested in a general overview of the distribution of mutations within structural superfamilies in cath, one can obtain a list of the superfamilies with known mutations and analyze domains in that superfamily.
once the user has selected a cath domain, 3dsim displays both an interactive jmol plug-in that allows the visualization of the mutations projected onto the three-dimensional structure of the representative cath domain and a table displaying all the information available for that given domain in terms of available mutations, sequence and structure positions of the mutations, pathogenicity information, and similarity  between the sequences in gene3d and the representative cath domain sequence.
this similarity index provides the user with a hint about the reliability of the homology based transference of mutations between sequences in gene3d and the structures in cath.
as a rule of thumb, the higher the similarity the more reliable the transference of mutations is.
tweaking this index is of particular interest when there are few mutations in the close relatives for a given structural family and looser constrains need to be taken into account to allow more mutations in the analysis.
by default, the server rejects those mutations transferred from sequences obtaining a blast sequence identity of less than 20%, but – due to the interactive approach of the server – the user can decide to establish more stringent constrains depending on the study case.
in addition, the site is linked to several external annotation providers  where more information about the mutations, the proteins and the structures can be gathered.
in particular, saapdb provides information about the structural implications of mutations.
this information can be related, in some cases, to the pathogenic character of the mutations and provides an insight into the mechanism of molecular function for several proteins.
description of web services
in order to allow remote programmatic access to the information contained in the database, we have developed a total of nine soap web services, powered by the perl soap::lite toolkit .
these allow users to retrieve in simple xml format:
• all known mutations for a given cath domain, grouped by uniprot id.
• the total number of mutations in a cath domain.
• all the cath domains which are associated with a given uniprot id.
• the amino-acid sequence of a given cath domain.
• all cath domains in a cath superfamily, queried by the four-part cath code.
• the superfamily to which a given cath domain belongs.
• the description and representative structure associated with a given cath superfamily.
• all the mutations in saapdb for a given uniprot accession.
• the total number of mutations in saapdb for a given uniprot accession.
these services were designed in such a way as to facilitate construction of computational analysis pipelines.
for example, a user starting with a uniprot protein of interest could retrieve a list of all the domains found in that protein, then the cath superfamilies to which each domain belongs, then all the other domains found in each superfamily, and filly all known mutations in those related domains, by chaining together four web service calls.
more information is provided at .
the page contains example perl code for querying the web-services, and examples of output from each one.
database update
the database storing the information presented by both the webserver and the webservice intrinsically depends on the other databases providing the source information , each one being updated at its own pace.
this fact, in addition to the computationally expensive calculations needed to compute the mapping between gene3d and the representative structures in cath, makes it impossible to schedule an automatic updating calendar.
therefore, the database will be updated based on a release system, where new versions will be made public as regularly as possible.
typical usage example
as an illustrative example, here we present the case of the atp binding subunit of the kinases  which is accessible through the server's web page .
this superfamily corresponds to the phosphotransferase domain i homology group in cath, and is subdivided into a number of different domains.
however, for this particular example, we will focus only on the domain with the highest number of mutations , 1rw8a <dig>  of these  <dig> mutations, only three come from the sequence which maps directly to the domain.
the remaining  <dig> come from homologous sequences with 40–65% sequence identity identified via gene3d .
figure  <dig> shows the structure with the pathogenic deviations coloured in blue.
this image can be obtained directly from the server, and is one of the main features available for the analysis of the distribution of mutations within structures.
additional links to other structure-based databases such as pdbsum  <cit>  are provided in order to enhance the information provided, for this particular case, the position of the catalytic site, involved in binding of atp, is described to be near residues from  <dig> to  <dig>  visual inspection of the position of the pathogenic deviations reveals that they tend to cluster around this catalytic core of the structure.
indeed, the higher the similarity in terms of blast identity between the cath domain and the gene3d sequence, the closer these positions are to the binding core and hence, more reliable the observations are.
this pdb chain  maps to the uniprotkb/swissprot accession p <dig> and the information provided by the uniprot record  shows that it corresponds to the tgf-beta receptor type- <dig> precursor in humans  for which there is a level of association with disease, in particular to furlong syndrome also known as loeys-dietz syndrome type 1a ;  <cit> .
lds <dig> is an aortic aneurysm syndrome with widespread systemic involvement.
the disorder is characterized by arterial tortuosity and aneurysms, craniosynostosis, hypertelorism, and bifid uvula .
other findings include exotropy, micrognathia and retrognathia, structural brain abnormalities, intellectual deficit, congenital heart disease, translucent skin, joint hyperlaxity and aneurysm with dissection throughout the arterial tree.
the mutations listed as pathogenic deviations  in the server for this protein, which has a 100% identity between the gene3d sequence and the representative structure of the cath domain are reported in the literature  <cit>  as involved in lds <dig> development.
CONCLUSIONS
we have presented 3dsim , a system that enables the localization and visualization of single amino acid polymorphisms projected onto protein structures based on homologous relationships captured in the cath and gene3d databases.
this provides a comprehensive overview of the distribution of mutations in structural space.
although there are other servers mapping mutations to structure already  the server presented here has several unique features not available in existing servers.
firstly, the similar treatment of snps and the rarer more harmful pds allows users to inspect and compare both kinds of mutation through the same interface, including explanatory metadata where available.
secondly, the localization of these saaps within the cath hierarchy allows users to query and explore the distribution of mutations at various levels of structural classification.
thirdly, the mapping of sequences onto homologous cath domains via gene3d helps users predict the effects of polymorphisms in proteins whose structure has not been solved.
finally, the availability of the data via web services and database dumps enables power users to include this information efficiently in their own analyses.
these facilities allow the independent integration of our data in any other pipeline or workflow.
the server has been running internally since we started working on the analysis of point mutations in protein families  <cit>  and is accessible at .
examples and documentation are also available, together with a tutorial video and samples of outputs of the main functions.
this website is available to all users with no login requirement.
it is likely that we will include additional features related with the structural interpretation of mutations and their relationship with disease, after receiving feedback from external users.
in summary the 3dsim server provides up-to-date, complete information automatically to map mutations in the domain sequences of proteins annotated in gene3d onto protein structures classified in the cath database.

 the most common biologically-relevant mutations are single base changes often referred to as single nucleotide polymorphisms. these account for about 90% of sequence polymorphisms in humans at an overall frequency of about one per bases. traditionally, snps are classified as coding or non-coding according to their genomic location – coding snps are further sub-classified according to the protein product expressed. non-synonymous snps are those that alter the amino acid sequence of the protein product, either through amino acid substitution, or by the generation of truncation mutations. by contrast, synonymous snps are those that do not alter the amino acid sequence of the protein product. not all synonymous snps are neutral since they may still affect the expression of gene products or protein translation by introducing alterations into regulatory regions, interfering with splice sites or impinging on other regulatory mechanisms. similarly, not all nssnps are associated with pathological diseases, since some changes are, by nature, milder than others, and diseases commonly involve complex sets of alterations. strictly the term'snp' is defined as a mutation which occurs in at least 1% of a'normal' population. thus snps are expected to have a neutral non-deleterious or low-penetrance phenotypic effect whereas the term pathogenic deviation refers to those mutations that generally occur at much lower frequencies in the population and have a severe effect on phenotype. the most commonly used database for storing information on snps is dbsnp, which currently contains several million validated snps from humans and other species. other sources of genomic information about snps include ensembl and the hapmap project. several efforts have been devoted to the prediction of the pathogenicity of amino acid mutations, resulting from single nucleotide changes. these methods make use of a set of characteristics which may be based both on sequence and structure, to determine whether a mutation can affect protein function and therefore be, potentially, associated with disease. this is an area of active research as shown by the considerable number of publications on the subject during the last few years. several efforts, saapdb among others, have been devoted to compiling this information and to providing a sequence and structural analysis, where possible, aiming to determine the origin of the pathogenicity shown. in this type of repository, the term snp is used to refer to essentially phenotypically silent mutations, while pd is used for mutations known to have a severe effect on phenotype, i.e. any single base change reported to correlate with disease. online mendelian inheritance in man is a collection of information about inherited disease and contains data on pds. however a great deal more information is held and maintained by individual research groups in locus-specific mutation databases or lsmdbs. like pds, nssnps are point mutations, but by definition they occur in at least 1% of a'normal' population. they are expected to have a neutral non-deleterious or low-pentrance phenotypic effect whereas pds are known to be detrimental. by mapping these saaps onto protein structures, we can begin to understand how protein structure might be affected by mutant residues, and so begin to explain the functional effect of the mutation. saapdb provides potential explanations for both pds, derived from various sources, and snps, derived from dbsnp. the cath structural domain database is a manually curated classification of domain structures found in the protein data bank, grouped according to evolutionary relationships and structural features. hidden markov models are derived from alignments of these structural exemplars and used by gene3d to identify homologues within the protein sequences of uniprot, refseq and ensembl. here we present 3dsim, a system mapping single amino-acid polymorphisms onto structures of cath domains. for sequences with no known structure, the gene3d resource of domain structure annotations is used to map the sequence onto the closest homologous domain of known structure in cath. thus 3dsim is of particular interest when no structural information is available for a protein in which mutations are known to occur as it uses sequence homology to map to the closest representative structure. this provides a comprehensive overview of the distribution of mutations in structural space, as well as a visualization tool for pinpointing the locations of mutations on individual structures rendered in jmol, as well as links to detailed information on each sequence, structure and mutation. the 3dsim application, which was designed with the aim of being very intuitive, easy to use and user-friendly, is publicly available at. several worked examples are available, along with a 6-minute video tutorial. in addition, for those advanced users needing intensive programmatic access to the information stored, the underlying database containing the mappings between saapdb, gene3d and cath is available on request, and most of the functionality is available as web services implemented in soap.  the mapping between saapdb and gene3d saapdb contains polymorphism data for sequences without a structure. almost all of these could be mapped to gene3d: identical sequences were found in the gene3d database. of the remaining had sequences with the same length and associated uniprot accession, leaving only for which a reliable match could not be obtained directly. the mapping between gene3d and cath where no structural data are available, the best representative cath domain is selected on the basis of homology. for each of the superfamilies in cath, a database of all cath domains was built. for each of the gene3d domain sequences mapped to cath structural superfamilies for which there is information about mutations in saapdb, a blast search was run against the corresponding superfamily database. the closest relative found was used to cluster the sequences. sequences with a sequence identity less than 20% were placed in separate clusters. this process yielded different groups. the groups were then aligned using muscle and the resulting alignments used to transfer the mutations from gene3d sequences to cath domain representative structures. at the end of the pipeline we were able to display information on point mutations, of them known to be associated with disease, mapping to cath superfamilies. the complete pipeline is described in figure and details are provided in the methods section. description of web application the initial input for the system is a cath superfamily identifier for which the user wants to retrieve information on mapped mutations. alternatively, the database can be searched using uniprot accession codes or cath domain identifiers. the user can either manually introduce the desired superfamily identifier in the provided form, or browse the superfamilies in cath in order to access the information. after this initial step, information about the selected cath superfamily is displayed along with the cath domains for which there is information about mutations in saapdb. in addition, for users that are interested in a general overview of the distribution of mutations within structural superfamilies in cath, one can obtain a list of the superfamilies with known mutations and analyze domains in that superfamily. once the user has selected a cath domain, 3dsim displays both an interactive jmol plug-in that allows the visualization of the mutations projected onto the three-dimensional structure of the representative cath domain and a table displaying all the information available for that given domain in terms of available mutations, sequence and structure positions of the mutations, pathogenicity information, and similarity between the sequences in gene3d and the representative cath domain sequence. this similarity index provides the user with a hint about the reliability of the homology based transference of mutations between sequences in gene3d and the structures in cath. as a rule of thumb, the higher the similarity the more reliable the transference of mutations is. tweaking this index is of particular interest when there are few mutations in the close relatives for a given structural family and looser constrains need to be taken into account to allow more mutations in the analysis. by default, the server rejects those mutations transferred from sequences obtaining a blast sequence identity of less than 20%, but – due to the interactive approach of the server – the user can decide to establish more stringent constrains depending on the study case. in addition, the site is linked to several external annotation providers where more information about the mutations, the proteins and the structures can be gathered. in particular, saapdb provides information about the structural implications of mutations. this information can be related, in some cases, to the pathogenic character of the mutations and provides an insight into the mechanism of molecular function for several proteins. description of web services in order to allow remote programmatic access to the information contained in the database, we have developed a total of nine soap web services, powered by the perl soap::lite toolkit. these allow users to retrieve in simple xml format: • all known mutations for a given cath domain, grouped by uniprot id. • the total number of mutations in a cath domain. • all the cath domains which are associated with a given uniprot id. • the amino-acid sequence of a given cath domain. • all cath domains in a cath superfamily, queried by the four-part cath code. • the superfamily to which a given cath domain belongs. • the description and representative structure associated with a given cath superfamily. • all the mutations in saapdb for a given uniprot accession. • the total number of mutations in saapdb for a given uniprot accession. these services were designed in such a way as to facilitate construction of computational analysis pipelines. for example, a user starting with a uniprot protein of interest could retrieve a list of all the domains found in that protein, then the cath superfamilies to which each domain belongs, then all the other domains found in each superfamily, and filly all known mutations in those related domains, by chaining together four web service calls. more information is provided at. the page contains example perl code for querying the web-services, and examples of output from each one. database update the database storing the information presented by both the webserver and the webservice intrinsically depends on the other databases providing the source information, each one being updated at its own pace. this fact, in addition to the computationally expensive calculations needed to compute the mapping between gene3d and the representative structures in cath, makes it impossible to schedule an automatic updating calendar. therefore, the database will be updated based on a release system, where new versions will be made public as regularly as possible. typical usage example as an illustrative example, here we present the case of the atp binding subunit of the kinases which is accessible through the server's web page. this superfamily corresponds to the phosphotransferase domain i homology group in cath, and is subdivided into a number of different domains. however, for this particular example, we will focus only on the domain with the highest number of mutations, 1rw8a of these mutations, only three come from the sequence which maps directly to the domain. the remaining come from homologous sequences with 40–65% sequence identity identified via gene3d. figure shows the structure with the pathogenic deviations coloured in blue. this image can be obtained directly from the server, and is one of the main features available for the analysis of the distribution of mutations within structures. additional links to other structure-based databases such as pdbsum are provided in order to enhance the information provided, for this particular case, the position of the catalytic site, involved in binding of atp, is described to be near residues from to visual inspection of the position of the pathogenic deviations reveals that they tend to cluster around this catalytic core of the structure. indeed, the higher the similarity in terms of blast identity between the cath domain and the gene3d sequence, the closer these positions are to the binding core and hence, more reliable the observations are. this pdb chain maps to the uniprotkb/swissprot accession p and the information provided by the uniprot record shows that it corresponds to the tgf-beta receptor type- precursor in humans for which there is a level of association with disease, in particular to furlong syndrome also known as loeys-dietz syndrome type 1a;. lds is an aortic aneurysm syndrome with widespread systemic involvement. the disorder is characterized by arterial tortuosity and aneurysms, craniosynostosis, hypertelorism, and bifid uvula. other findings include exotropy, micrognathia and retrognathia, structural brain abnormalities, intellectual deficit, congenital heart disease, translucent skin, joint hyperlaxity and aneurysm with dissection throughout the arterial tree. the mutations listed as pathogenic deviations in the server for this protein, which has a 100% identity between the gene3d sequence and the representative structure of the cath domain are reported in the literature as involved in lds development.  we have presented 3dsim, a system that enables the localization and visualization of single amino acid polymorphisms projected onto protein structures based on homologous relationships captured in the cath and gene3d databases. this provides a comprehensive overview of the distribution of mutations in structural space. although there are other servers mapping mutations to structure already the server presented here has several unique features not available in existing servers. firstly, the similar treatment of snps and the rarer more harmful pds allows users to inspect and compare both kinds of mutation through the same interface, including explanatory metadata where available. secondly, the localization of these saaps within the cath hierarchy allows users to query and explore the distribution of mutations at various levels of structural classification. thirdly, the mapping of sequences onto homologous cath domains via gene3d helps users predict the effects of polymorphisms in proteins whose structure has not been solved. finally, the availability of the data via web services and database dumps enables power users to include this information efficiently in their own analyses. these facilities allow the independent integration of our data in any other pipeline or workflow. the server has been running internally since we started working on the analysis of point mutations in protein families and is accessible at. examples and documentation are also available, together with a tutorial video and samples of outputs of the main functions. this website is available to all users with no login requirement. it is likely that we will include additional features related with the structural interpretation of mutations and their relationship with disease, after receiving feedback from external users. in summary the 3dsim server provides up-to-date, complete information automatically to map mutations in the domain sequences of proteins annotated in gene3d onto protein structures classified in the cath database. 
these polymorphisms are drawn from saapdb, which aggregates data from various sources including dbsnp and several pathogenic mutation databases. this resource contains sequences annotated with domains predicted to belong to structural families in the cath database. here we present 3dsim, a database and web application facilitating the localization and visualization of single amino acid polymorphisms mapped to protein structures even where the structure of the protein of interest is unknown. mappings between domain sequences in gene3d and known structures in cath are obtained using a muscle alignment. three-dimensional structures corresponding to cath structural domains are currently included in 3dsim; these domains are distributed across cath superfamilies, and provide a comprehensive overview of the distribution of mutations in structural space.
 the phenotypic effects of sequence variations in protein-coding regions come about primarily via their effects on the resulting structures, for example by disrupting active sites or affecting structural stability. in order better to understand the mechanisms behind known mutant phenotypes, and predict the effects of novel variations, biologists need tools to gauge the impacts of dna mutations in terms of their structural manifestation. although many mutations occur within domains whose structure has been solved, many more occur within genes whose protein products have not been structurally characterized.  here we present 3dsim, a database and web application facilitating the localization and visualization of single amino acid polymorphisms mapped to protein structures even where the structure of the protein of interest is unknown. the server displays information on point mutations, of them known to be associated with disease. these polymorphisms are drawn from saapdb, which aggregates data from various sources including dbsnp and several pathogenic mutation databases. while the saapdb interface displays mutations on known structures, 3dsim projects mutations onto known sequence domains in gene3d. this resource contains sequences annotated with domains predicted to belong to structural families in the cath database. mappings between domain sequences in gene3d and known structures in cath are obtained using a muscle alignment. three-dimensional structures corresponding to cath structural domains are currently included in 3dsim; these domains are distributed across cath superfamilies, and provide a comprehensive overview of the distribution of mutations in structural space.  the server is publicly available at. in addition, the database containing the mapping between saapdb, gene3d and cath is available on request and most of the functionality is available through programmatic web service access. eccb workshop: annotations, interpretation and management of mutations cagliari, italy 