the internal transcribed spacer 2 ( its2 ) of the nuclear rrna cistron is a widely used phylogenetic marker . as its sequence evolves comparably fast , it is mainly used for low - level analyses . the hallmarks , namely four helices with the third as the longest , have been found in detailed exemplary studies ( 1 ) as well as in large - scale analyses ( 2 ) . this lead to the suggestion to enlarge the application field to higher taxonomic levels ( 3 ) . in addition to these phylogenetic analyses , a specific structural feature between two its2 , a compensatory base change ( cbc ) , can be used to distinguish two species from each other ( 4 ) . this underlines the importance of considering not only the sequence but also the structure when performing any analysis based on the its2 . but the proposed correct structure is only rarely automatically found by standard minimum free energy folding ( mfe ) ( 2 ) . to overcome this hindrance for the wider application of the its2 , we developed a homology - based structure modelling approach , which allowed predicting the structure for 20 000 sequences which were not found by rnafold ( 5 ) . as these can be used as a basis for any phylogenetic analysis , we have developed the its2 database as a resource for sequence and structure information of the its2 ( 6 ) . here we report modifications and improvements of the database which allowed us to find structural information for 86 784 its2 sequences , which is 55% of all entries concerning its2 in genbank . in the first version of the database , every sequence whose correct structure could not be found by rnafold was searched against the original set of 5 000 sequences with correct rnafold based structures ( 2 ) to identify possible templates for homology modelling ( models ) . as a first step in the development of the new version of the database , we checked whether there were additional novel sequences in genbank whose structure could be determined directly by rnafold . indeed , we found a 2-fold increase in the amount of correctly predicted structures ( table 1 , method 1 ) . we used this dataset as a starting point for a complete rebuild of the database . more importantly , this result led us to a change in the logic and therefore to a re - design of the update procedure . each time the structure of an incoming sequence can be predicted directly by rnafold or in the first round of homology modelling , it is added to the set of models . thus , no core sequence / structure set ( as before the set of 5 000 ) is existent any more but a dynamically growing set of possible structure models . in summary , this approach together with a second iteration of homology modelling allowed us to predict 38 350 structures ( table 1 , methods 2 and 3 ) . table 1.methods used for its2 structure prediction and number of folded sequences.methoddescriptioncount1direct rnafold10 6672homology modelling , first iteration27 0443homology modelling , second iteration11 3064direct rnafold , sequence discovery by blast5 1965homology modelling , first iteration , sequence discovery by blast1 7306homology modelling , second iteration , sequence discovery by blast17 7767partial structures from homology modelling , both iterations13 065total86 784 methods used for its2 structure prediction and number of folded sequences . if the boundaries are incorrect , missing or additional sequence fragments might be considered as a specific feature of an organism leading to a wrong phylogenetic classification . with the correct structure at hand , the boundaries of the its2 can be exactly determined , again underlining the importance of considering structure for phylogenetic analyses . accordingly , already in the first version of the database , a clustalw - based approach ( 7 ) was used to extend the sequence if the genbank annotation missed the first or the last helix . as this approach was limited to cases where ( i ) there exists a feature annotation by genbank and ( ii ) the homology modelling was of high quality for the other helices , we developed a novel , blast - based approach ( 8) for the re - annotation of genbank entries . for the cases where no structure could be predicted for the its2 as annotated by genbank , the whole genbank entry is retrieved and searched against all sequences with known structures using blast . if a significant hit is found ( e - value 10e ) , the homologous region of the query is cut . rnafold is used to test whether this fragment can be folded in the correct structure . if not , we were able to re - annotate the position of the its2 in 6901 genbank entries . in most cases , this structure - based annotation lead to a slight shift of the 5 ' or / and the 3 ' end of the its2 , but some entries were heavily shifted ( figure 1 ) . for example the its2 of trifolium affine ( gi:85724133 ) is incorrectly annotated with a length of just 7 bp , its preceding 5.8s ribosomal rna with 9 bp . these cases underline the advantage of the structure - based annotation compared to one based on sequence information alone . figure 1.re-annotated sequences , each dot representing a successfully predicted secondary structure  x - axis represents shift in the 5 ' end of the its2 , y - axis change of the length compared to the genbank annotation . six outliers ( gi : 5814072 , 57999795 , 2896060 , 13507073 , 4006937 , 85724147 ) are not shown . re - annotated sequences , each dot representing a successfully predicted secondary structure  x - axis represents shift in the 5 ' end of the its2 , y - axis change of the length compared to the genbank annotation . six outliers ( gi : 5814072 , 57999795 , 2896060 , 13507073 , 4006937 , 85724147 ) are not shown . in contrast to the method used in the previous version of the database , the blast - based approach is completely independent of any pre - annotated its2 . its2 without a feature annotation lead to the new annotation of 17 801 its2 sequences . many of the sequences without predicted structure were fragments , i.e. they missed at least one helix of the structural hallmark and therefore did not fulfil the quality control of the standard homology modelling . still , these sequences could increase the coverage of a systematic analysis . in contrast to the mfe approach , our homology - modelling algorithm is able to predict the structure of fragments . to assure a sufficient quality , only entries where at least two consecutive helices could be modelled with sufficient quality ( 75% ) were accepted . this method resulted in additional 13 065 its2 sequences with structural information ( table 1 , method 7 ) . the existence of a large number of pairwise alignments allowed us to calculate its2-specific evolutionary models . based on variants of the methods described in mller and vingron ( 9 ) and mller et al . ( 10 ) , we were able to derive an its2-specific substitution model , which is an important ingredient for phylogenetic analyses . this model reflects nicely the special features of rna and in particular , its2 sequence evolution . based on this molecule - specific substitution model , an its2-specific scoring matrix is derived that strongly deviated from the unity matrix as used as default , for example in blast . to test the influence of this matrix compared to the standard identity matrix , we performed all calculations with the standard and the its2-specific matrix , respectively . in the genbank version used in the test run , structural information was found for 57 680 sequences whereas the usage of the its2-specific matrix resulted in 76 721 structures . this underlines the importance of the correct evolutionary model in the homology modelling of its2 and presumably other rna sequences . accordingly , the its2-specific score matrix is now used in all calculations for the its2 database and can be downloaded from the web site as supplementary data . here , the user provides an rna sequence with a known structure and other , homologous sequences . for these , a homology model is calculated based on the known structure . when using this feature , it has to be taken into account that there is , in contrast to the modelling of its2 , no quality measure for the model . thus , it is the obligation of the user to check the validity of the results . in the first version of the database , every sequence whose correct structure could not be found by rnafold was searched against the original set of 5 000 sequences with correct rnafold based structures ( 2 ) to identify possible templates for homology modelling ( models ) . as a first step in the development of the new version of the database , we checked whether there were additional novel sequences in genbank whose structure could be determined directly by rnafold . indeed , we found a 2-fold increase in the amount of correctly predicted structures ( table 1 , method 1 ) . we used this dataset as a starting point for a complete rebuild of the database . more importantly , this result led us to a change in the logic and therefore to a re - design of the update procedure . each time the structure of an incoming sequence can be predicted directly by rnafold or in the first round of homology modelling , it is added to the set of models . thus , no core sequence / structure set ( as before the set of 5 000 ) is existent any more but a dynamically growing set of possible structure models . in summary , this approach together with a second iteration of homology modelling allowed us to predict 38 350 structures ( table 1 , methods 2 and 3 ) . table 1.methods used for its2 structure prediction and number of folded sequences.methoddescriptioncount1direct rnafold10 6672homology modelling , first iteration27 0443homology modelling , second iteration11 3064direct rnafold , sequence discovery by blast5 1965homology modelling , first iteration , sequence discovery by blast1 7306homology modelling , second iteration , sequence discovery by blast17 7767partial structures from homology modelling , both iterations13 065total86 784 methods used for its2 structure prediction and number of folded sequences . if the boundaries are incorrect , missing or additional sequence fragments might be considered as a specific feature of an organism leading to a wrong phylogenetic classification . with the correct structure at hand , the boundaries of the its2 can be exactly determined , again underlining the importance of considering structure for phylogenetic analyses . accordingly , already in the first version of the database , a clustalw - based approach ( 7 ) was used to extend the sequence if the genbank annotation missed the first or the last helix . as this approach was limited to cases where ( i ) there exists a feature annotation by genbank and ( ii ) the homology modelling was of high quality for the other helices , we developed a novel , blast - based approach ( 8) for the re - annotation of genbank entries . for the cases where no structure could be predicted for the its2 as annotated by genbank , the whole genbank entry is retrieved and searched against all sequences with known structures using blast . if a significant hit is found ( e - value 10e ) , the homologous region of the query is cut . rnafold is used to test whether this fragment can be folded in the correct structure . if not , we were able to re - annotate the position of the its2 in 6901 genbank entries . in most cases , this structure - based annotation lead to a slight shift of the 5 ' or / and the 3 ' end of the its2 , but some entries were heavily shifted ( figure 1 ) . for example the its2 of trifolium affine ( gi:85724133 ) is incorrectly annotated with a length of just 7 bp , its preceding 5.8s ribosomal rna with 9 bp . these cases underline the advantage of the structure - based annotation compared to one based on sequence information alone . figure 1.re-annotated sequences , each dot representing a successfully predicted secondary structure  x - axis represents shift in the 5 ' end of the its2 , y - axis change of the length compared to the genbank annotation . six outliers ( gi : 5814072 , 57999795 , 2896060 , 13507073 , 4006937 , 85724147 ) are not shown . re - annotated sequences , each dot representing a successfully predicted secondary structure  x - axis represents shift in the 5 ' end of the its2 , y - axis change of the length compared to the genbank annotation . six outliers ( gi : 5814072 , 57999795 , 2896060 , 13507073 , 4006937 , 85724147 ) are not shown . in contrast to the method used in the previous version of the database , the blast - based approach is completely independent of any pre - annotated its2 . its2 without a feature annotation lead to the new annotation of 17 801 its2 sequences . many of the sequences without predicted structure were fragments , i.e. they missed at least one helix of the structural hallmark and therefore did not fulfil the quality control of the standard homology modelling . in contrast to the mfe approach , our homology - modelling algorithm is able to predict the structure of fragments . to assure a sufficient quality , only entries where at least two consecutive helices could be modelled with sufficient quality ( 75% ) were accepted . this method resulted in additional 13 065 its2 sequences with structural information ( table 1 , method 7 ) . the existence of a large number of pairwise alignments allowed us to calculate its2-specific evolutionary models . based on variants of the methods described in mller and vingron ( 9 ) and mller et al . ( 10 ) , we were able to derive an its2-specific substitution model , which is an important ingredient for phylogenetic analyses . this model reflects nicely the special features of rna and in particular , its2 sequence evolution . based on this molecule - specific substitution model , an its2-specific scoring matrix is derived that strongly deviated from the unity matrix as used as default , for example in blast . to test the influence of this matrix compared to the standard identity matrix , we performed all calculations with the standard and the its2-specific matrix , respectively . in the genbank version used in the test run , structural information was found for 57 680 sequences whereas the usage of the its2-specific matrix resulted in 76 721 structures . this underlines the importance of the correct evolutionary model in the homology modelling of its2 and presumably other rna sequences . accordingly , the its2-specific score matrix is now used in all calculations for the its2 database and can be downloaded from the web site as supplementary data . here , the user provides an rna sequence with a known structure and other , homologous sequences . for these , a homology model is calculated based on the known structure . when using this feature , it has to be taken into account that there is , in contrast to the modelling of its2 , no quality measure for the model . thus , it is the obligation of the user to check the validity of the results . with the modifications of the its2 database outlined above , the structural features of 86 784 sequences were predicted , which was 55% of all genbank entries concerning the its2 . as this number gives just an overall average , we tested the coverage of predicted structures within all genera . a clear separation was found between genera where the structure for nearly all sequences could be predicted and others , where no structure was found despite considerable sequence coverage ( figure 2 ) . we suggest that in these genera the structure of the its2 deviates from the standard . this notion is supported by their length distribution being nearly equal to the length distribution of successfully folded sequences ( data not shown ) . furthermore , within those genera without any structural data , there is a strong bias towards metazoans ( 11 ) . this is consistent with the observation that vertebrates have a more complex structure than the one described by coleman ( 1 ) . the latter one fits mostly for plants and fungi , taxa whose genera are strongly represented in the overall number of genera with structural data . figure 2.structure coverage  each point indicates one genus . on the y - axis , the square root of the number of sequences in the genus is indicated . on the x - axis , the percentage of correct structures for all sequences of the genus is plotted . additionally on top of the scatter plot , a similar , less pronounced effect can be seen at 33.3% and 66.6% for genera with three sequences . the square root of the number of sequences in the genus is indicated . on the x - axis , the percentage of correct structures for all sequences of the genus is plotted . additionally on top of the scatter plot , a similar , less pronounced effect can be seen at 33.3% and 66.6% for genera with three sequences . how could a user , who is interested in the phylogeny of a specific taxonomic group , use the its2 database ? if he starts with an already known sequence , he can directly extract the corresponding structure from the database (  search by gi / accession / taxon ) . if he has sequenced his own organisms , he should first homology model the structure of this sequences (  predict its2 structure ) . second , he can extract its2 sequences and their structures for further organisms in the taxonomic group of interest (  browse taxonomy ) . this will result in a set of its2 sequences with corresponding structures . in the third step , these have to be aligned . here , an alignment program , which considers both sequence and structure , like 4sale ( 12 ) , will be suitable . finally , this sequence  structure - based alignment will be the input for standard phylogenetic analyses , e.g. in paup ( 13 ) or phylip ( 14 ) . furthermore , one is now able to check for cbcs to distinguish possible different species in the dataset ( 4 ) or to calculate cbc trees ( 15 ) .
an increasing number of phylogenetic analyses are based on the internal transcribed spacer 2 ( its2 ) . 
 they mainly use the fast evolving sequence for low - level analyses . 
 when considering the highly conserved structure , the same marker could also be used for higher level phylogenies . furthermore 
 , structural features of the its2 allow distinguishing different species from each other . despite its importance , 
 the correct structure is only rarely found by standard rna folding algorithms . to overcome this hindrance for a wider application of the its2 , 
 we have developed a homology modelling approach to predict the structure of rna and present the results of modelling the its2 in the its2 database . here , we describe the database and the underlying algorithms which allowed us to predict the structure for 86 784 sequences , which is more than 55% of all genbank entries concerning the its2 . 
 these are not equally distributed over all genera . 
 there is a substantial amount of genera where the structure of nearly all sequences is predicted whereas for others no structure at all was found despite high sequence coverage . 
 these genera might have evolved an its2 structure diverging from the standard one . 
 the current version of the its2 database can be accessed via http://its2.bioapps.biozentrum.uni-wuerzburg.de .