during recent years , there has been an increasing interest in the structural features of protein phosphorylation sites ( p - sites ) . this fact can be ascribed to the steadily growing experimentally verified p - sites provided by high - throughput mass spectrometry - based proteomics techniques [ e.g. ( 1 ) ] . the simultaneous availability of an increasing number of three - dimensional ( 3d ) structures is making it possible to infer the structural context for a significant number of p - sites . in order to identify the structural determinants of kinase specificity , some authors tried to characterize the 3d environment of p - sites with the aim of pinpointing specific structural features ( 24 ) . both phosphorylation sites databases , also reporting structural data , and interesting systematic structural analyses of p - sites have recently appeared in the literature [ for a review , see ref . phosida ( 1 ) , a phosphorylation sites database , includes the predicted accessibility and secondary structure of each p - site . the mtcptm ( 6 ) database stores homology models for proteins and protein domains that contain phosphorylated residues . finally , many of the p - site predictors incorporating 3d - context information ( 1,4,710 ) display an improvement in performance with respect to predictors using sequence information only . so far the structural attributes stored in p - site databases and incorporated in p - site predictors are essentially of two types : accessibility and secondary structure . in 2007 , we presented phospho3d , a database of reliably predicted 3d structures of protein phosphorylation sites ( 11 ) , derived from the phospho.elm database ( 12 ) and enriched with structural annotation at the residue level , including accessibility , secondary structure and residue conservation as from the consurf - hssp database ( 13 ) . phospho3d also stored and annotated the sequence flanking the p - site ( 10 residues ) and the zone , i.e. the 3d region defined by the set of residues at a distance not exceeding 12   from the phospho - instance . since then , the number of phospho.elm instances increased 4-fold , raising from 5314 ( in 1805 proteins ) to 42  474 ( in 8718 proteins ) , and more than 26  000 structures were added to the protein data bank ( pdb ) ( 14 ) . here we introduce phospho3d version 2.0 , which  besides an eleven - fold increase in the number of phospho.elm unique instances mapped onto 3d structures ( compared to version 1.0)incorporates several new features , including additional structural descriptors of p - sites , the possibility of browsing the database selecting non - redundant sets of 3d structures , the availability for download of many non - redundant sets of structurally annotated p - sites  aimed at serving as reliable benchmark datasets for predictors training and test  and p3dscan , a new functionality that allows the user to submit a protein structure and scan it against the 3d p - site zones collected in the phospho3d database . the updated phospho3d database was constructed by collecting data from the latest release of the phospho.elm database ( version 9.0 , august 2010 ) , which currently stores about 42  500 experimentally verified phosphorylation sites in 8718 substrate proteins , both manually extracted from the literature and obtained from mass spectrometry - based proteomics experiments . the correspondence between phospho.elm sequences and pdb chains was based on sequence alignment using at least 98% sequence identity . this resulted in 5387 mapped instances ( 1770 unique phospho.elm instances on 2158 protein chains897 ser , 338 thr , 535 tyr ) . notice that p - sites derived from mass spectrometry ( ms ) experiments should be taken with caution . in fact , due to the current procedures for ms data deposition , it is difficult to systematically detect if a phospho - instance was identified in physiologically abnormal conditions ( e.g. in proteins extracted from oncogenic tissues or that do not undergo phosphorylation , such as hemoglobin ) ( 15 ) . in order to help users detect such potentially problematic cases , we reported  for each p - site  the nature of the original experiment ( low- or high - throughput ) and the corresponding literature reference ( pmid ) . moreover , we encourage users to carefully analyze the structural context of p - sites , which might be indicative of problems in the original data . one example is represented by the tyr phosphorylation site mapped to position 133 of the human hemoglobin subunit beta ( uniprotkb : p68871 ) , for which phospho3d stores 43 pdb structures . in most of the reported structures , the basic information stored in phospho3d consists of the p - site instance , its flanking sequence ( 10 residues ) and the p - site 3d zone , i.e. the set of residues in a 12   radius surrounding the p - site in space . for each residue in the zone phospho3d 2.0 stores the following structural descriptors : secondary structure and solvent accessibility ( in  ) as defined by dssp ( 16 ) ; percentage solvent accessibility , obtained by normalizing the dssp solvent accessibility by the maximum accessibility value for each residue as determined in ref . ( 17 ) ; b - factor , computed as specified in ref . ( 18 ) ; occurrence in a cavity together with the rank and volume of the cavity calculated with the surfnet program ( 19 ) ; the depth index dpx ( 20 ) and the protrusion index cx ( 21 ) , obtained using the psaia software ( 22 ) ; the consurf conservation score extracted from consurfdb ( 23 ) ; the disorder probability provided by disembl ( 24 ) according to three different criteria : ( i ) loops / coils as defined by dssp , ( ii ) hot loops , i.e. loops with a high degree of mobility as determined by temperature ( b- ) factor and ( iii ) missing coordinates in x - ray structure as defined by remark-465 entries in pdb . phospho3d 2.0 also provides information derived from phospho.elm , such as , when available , the kinase(s ) phosphorylating a given p - site , and , for each zone , the results of a large - scale local structural comparison versus a non redundant ( sequence identity 20% ) dataset of 487 pdb x - ray protein chains with experimental resolution 1.5   extracted from eukaryotic organisms . the comparison is carried out using the new version of the algorithm ( 25 ) and the same criteria for assessing structural similarity used in the previous database version ( 11 ) although more stringent thresholds are applied in this case , as described in the website documentation . the database queries can now be performed on seven pdb non - redundant sets : the whole collection of p - sites , the set of p - sites found in non - identical structures ( pdb100 ) and p - sites found in pdb structures belonging to five redundancy sets , ranging from pdb90 to pdb20 , where the number corresponds to the maximum sequence identity shared by the protein chains in the redundancy set . these sets have been determined using the pisces resource ( 26 ) . additionally , the p - site annotations at the residue level are available for download on the phospho3d website . these can serve as benchmark for p - site predictors training and test and for analyses of p - site structural features . finally , phospho3d 2.0 now links each entry to the corresponding phospho.elm instance and the kinase names to their uniprot acs ( 27 ) . phospho3d 2.0 provides a novel functionality that allows the user to upload a pdb - formatted structure and perform a local structural comparison against the 5387 zones ( one for each phospho.elm mapped instance ) stored in the database , aimed at identifying local structural similarities between the user query structure and one of the structural patches containing a p - site . in order to evaluate the structural context of each match , we provide its graphical display and a table reporting the structural information at the residue level of both the query and the target 3d matching patches . the comparison algorithm  that p3dscan runs on - the - fly  is the same as the one used for the large - scale comparison whose results are stored in the database . phospho3d 2.0 provides a novel functionality that allows the user to upload a pdb - formatted structure and perform a local structural comparison against the 5387 zones ( one for each phospho.elm mapped instance ) stored in the database , aimed at identifying local structural similarities between the user query structure and one of the structural patches containing a p - site . in order to evaluate the structural context of each match , we provide its graphical display and a table reporting the structural information at the residue level of both the query and the target 3d matching patches . the comparison algorithm  that p3dscan runs on - the - fly  is the same as the one used for the large - scale comparison whose results are stored in the database . similarly to the previous version , phospho3d 2.0 can be searched by kinase name , by pdb identification code or by keyword . in this new version , however , the user can additionally select a redundancy set in order to avoid retrieving identical or very similar p - sites . the data returned to the user consist of a brief description of the pdb structure(s ) that fulfill the search criteria and a list of instances presented along with associated information . in particular , each instance is now linked to the corresponding phospho.elm entry . for each p - site , the user can select three options related to the surrounding structural zone : a graphical view using the jmol java applet ( http://www.jmol.org ) , a tabular view reporting the zone annotation at the residue level or a list of 3d matches identied by local structural comparison . users upload a pdb - formatted file , choose a redundancy set of 3d zones they want to scan against their structure and run the comparison by clicking the can be graphically visualized by clicking on the corresponding button . moreover , the tabular view button links to a window displaying structural annotation at the residue level , both for the query and the target 3d patches . figure 1.the p3dscan output page for the crystal structure of the h - ras oncogene protein p21 ( pdb 5p21 ) . the table reports the list of matches between the query protein ( probe ) and the zone(s ) collected in the database ( target ) . scop and cath annotation , when available , is reported for both the query structure and for each matching zone therefore the user can discriminate matches due to overall fold similarity from functionally interesting  ones . the 3d zone ( column 5 ) is linked to the corresponding phospho3d database entry and the phospho - residue is explicitly reported in column 6 . the score ( column 9 ) corresponds to the number of paired residues in the match . upper right inset : graphical display ( jmol ) of the probe and target structures . residues participating in the match are in stick and the p - site is colored in orange . bottom inset : tabular view : structural information at the residue level is reported for both the probe ( user query structure ) and target ( 3d zone ) residues involved in the match . the p3dscan output page for the crystal structure of the h - ras oncogene protein p21 ( pdb 5p21 ) . the table reports the list of matches between the query protein ( probe ) and the zone(s ) collected in the database ( target ) . scop and cath annotation , when available , is reported for both the query structure and for each matching zone therefore the user can discriminate matches due to overall fold similarity from functionally interesting  ones . the 3d zone ( column 5 ) is linked to the corresponding phospho3d database entry and the phospho - residue is explicitly reported in column 6 . the score ( column 9 ) corresponds to the number of paired residues in the match . upper right inset : graphical display ( jmol ) of the probe and target structures . residues participating in the match are in stick and the p - site is colored in orange . bottom inset : tabular view : structural information at the residue level is reported for both the probe ( user query structure ) and target ( 3d zone ) residues involved in the match . we performed a large - scale structural analysis of the p - sites stored in phospho3d and plotted the statistical distributions of each 3d attribute used to annotate the p - sites in the database . the distributions for the p - sites falling on non - identical structures ( pisces pdb100 ) can be found at http://www.phospho3d.org/stats.py#3 . the new version of phospho3d stores a markedly increased number of structurally annotated p - sites . in addition , it incorporates new significant improvements , such as several new structural descriptors , non - redundant datasets and a tool , p3dscan , for the analysis of uploaded protein structures . we believe that this enhanced version of the database makes it possible to fully exploit available structural information on p - sites and use it to perform structural analyses and/or build p - site predictors . importantly , the phospho3d update procedure is now completely automated , allowing regular and timely updates of the database with each new phospho.elm release . fondazione cenci bolognetti , roma ; the 7th ec framework programme leishdrug project [ grant number 223414 ] ; and a  juan de la cierva fellowship to a.z .
phospho3d is a database of three - dimensional ( 3d ) structures of phosphorylation sites ( p - sites ) derived from the phospho.elm database , which also collects information on the residues surrounding the p - site in space ( 3d zones ) . 
 the database also provides the results of a large - scale structural comparison of the 3d zones versus a representative dataset of structures , thus associating to each p - site a number of structurally similar sites . 
 the new version of phospho3d presents an 11-fold increase in the number of 3d sites and incorporates several additional features , including new structural descriptors , the possibility of selecting non - redundant sets of 3d structures and the availability for download of non - redundant sets of structurally annotated p - sites . 
 moreover , it features p3dscan , a new functionality that allows the user to submit a protein structure and scan it against the 3d zones collected in the phospho3d database . 
 phospho3d version 2.0 is available at : http://www.phospho3d.org/.