protein structure comparison plays an essential role in understanding the similarities and differences between proteins , locating distantly related homologs / analogs , revealing functionality from similarity , and elucidating the often cryptic biological role in metabolic pathways .
protein comparison is both a complex and multidimensional problem , and while there are numerous structural alignment programs , structural comparison remains an active area of research .
a review is outside of the scope of this paper and reviews can be found elsewhere .
presently , there is a vast and rapidly growing quantity of publicly available protein structures , with the number of structures dramatically increasing since the advent of the structural genomics initiative ( 1 ) .
currently , one - to - all alignments to all other available structures must be calculated when a researcher is interested in a relation of a particular protein , be it published or private .
the task becomes even more challenging when an analysis of all - to - all relations is required , for instance in structural classification or functional annotation . and
while both computer speeds and heuristics have decreased the amount of time needed for such calculations , the sheer quantity of alignments ( 2 512 578 783 ) and size of data make such a calculation cumbersome ( estimate based on 74 613 chains as of july 25 , 2006 ) .
therefore , there are needs to have pre - calculated datasets of structural alignments between representative protein structures available to researchers for quick and easy access by request from a public database .
although currently there are several popular protein structural alignment databases ( 214 ) there is still no uniformly accepted gold standard ; moreover the alignment methods behind the databases produce different results , for example , the fssp and ce databases overlap in only 40% of the cases ( 15 ) .
additionally , a recent study found the best alignment , coined  best - of - all from a combination of six methods , is missed in 1050% of the cases by many of the commonly used methods ( 16 ) .
furthermore , the attempts to classify protein structures into hierarchical levels , albeit extremely functional and useful , do face an emerging alternative view in which protein space is not uniformly discrete , but more continuous and multidimensional ( 17 ) .
one such study has shown similarities between folds belonging to different levels of classification , bringing into question whether or not a fold designation should be viewed too rigidly ( 18 ) . with this alternative point existing , along with the lack of support establishing 
one structural alignment method as the most effective for all alignment pairs , we have developed topofit - db ( t - db ) .
t - db is a public web - based relational database of structural alignments based on our recently developed topofit method ( 19 ) .
the approach in which topofit alignments are produced is considerably different from the alignments produced by other popular methods .
the majority of methods attempt to balance between lower rmsd ( root mean square deviation ) and a higher number of aligned positions ( ne ) .
the approach implemented in the topofit method employs a different strategy , one in which equivalent nearest neighbors are exploited instead of better fit .
topofit identifies the largest group of residues which have the same neighbors in the same locations common in both compared structures , defined mathematically as a topological invariant .
the nearest neighbors have been defined uniquely for each structure by simple , well known delaunay tessellation ( dt ) ( 20 ) .
therefore , topofit does not use any heuristics based on rmsd , gap penalty , or alignment length ( ne ) or any combination of the three as input parameters to produce alignments .
the procedure is reversed : first a saturation point in the spatial tessellation graph is detected ( topomax point ) and then the corresponding c atoms and corresponding values between the aligned structures are reported .
such an objective methodology provides unambiguous identification and separation of the structurally invariant parts from the variable parts by identifying a precise border between the two .
one of the intrinsic values of the topofit method is the ability to produce non - sequential alignments , from circular permutations to complex and completely reverse alignments .
many single examples of proteins with non - sequentially aligned regions have been reported ( 21,22 ) ; therefore , the ability to determine non - sequential alignments will permit more extensive analysis of core protein structure topology .
topofit has been integrated into the friend software ( 23 ) and is capable of reproducing and visualizing alignments stored in t - db . to assist in the corroboration of a non - sequential alignment
the user can visualize the corresponding alignment plot ( figure 1c ) , and display the structural superimposition in the friend applet or stand alone application .
figure 1 displays the retrieved data from t - db , along with the structural superimposition and the corresponding alignment plot from the alignment of human frataxin ( pdb - code  1ekg chain a ) and hypothetical protein tm1457 ( pdb - code  1s12 chain c ) . a precise structural match ( rmsd < 2  )
expands almost entirely over both polypeptide chains with the alignment consisting of four fragments , with three fragments aligned in reverse order .
it should be mentioned , this structural relation is not present in existing structural alignments databases .
topofit is capable of calculating non - sequential alignments since the method does not rely on backbone extension to produce structural alignments , i.e. segments do not have to be sequentially ordered .
( a ) initial output page ( web - browser ) shown from a search using protein 1s12 chain c , all values from t - db are shown in table format , by selecting the 
align button , a new browser is opened showing the alignment plot , sequence alignment , and initializing the friend applet .
( b ) friend applet demonstrating the topofit superimposition ( backbone representation ) between human frataxin ( pdb - code  1ekg chain a , blue ) and hypothetical protein tm1457 ( pdb - code  1s12 chain c , green ) , ne 74 , rmsd 1.8  .
( c ) an example of a non - sequential alignment found by the topofit method , the alignment plot between 1ekg chain a ( x - axis ) and 1s12 chain c ( y - axis ) is shown , notice the three reverse fragments ( dotted circles ) . using the topofit method ,
we have developed the topofit database ( t - db ) for public use ; along with t - server for one - to - all comparisons with known structures from the pdb , and t - pair for the comparison of any two protein structures . to provide users with an effective way to utilize the data from t - db ,
this software package allows a user to conveniently and simultaneously visualize , and analyze multiple structural superimpositions and sequence alignments .
the software package includes both an applet for straightforward online viewing of the structure - sequence alignments , and a stand - alone version , which must be locally installed .
t - db release ( 0.9 ) currently contains 86 033 950 topofit structural alignments , 66 161 pdb protein chains and 9209 representatives ( centroids ) from the pdb ( 24 ) ( july 2005 ) .
the database has been initiated using the ce clusters of the structural neighbors from the ce database ( february 2002 release ) ( generously provided by dr i. shindyalov ) .
protein structures inside each ce cluster have been aligned by topofit all - to - all and a new representative structure has been chosen by the criteria of maximum sum of z - scores to all other structures in the cluster and named 
all the centroids have been compared to each other by topofit and the clusters with ne ( < 15 for up to 100 residues , and < 30 for over 100 residues in protein length ) between centroids have been joined together resulting in 3579 initial clusters .
a calculation pipeline using simple assignment procedure has been developed to automatically add new structures to the clusters .
each new structure has been compared to all other centroids using the same criteria as above : if a close relation to a centroid is present the structure was assigned to it , if not then a new cluster has been created and added to the list of centroids used for comparison there after , resulting in 9208 centroids .
this stringent clustering has resulted in tight clusters , where all the members of the cluster are essentially the same , which is important when a search is initiated in t - db , because centroids are used in all searching in an attempt to circumvent the high degree of redundancy found throughout the pdb .
the calculations have been performed on two clusters : our in - house 20 node dual cpu 2.8 ghz cluster and neu 's 65 node dual 3.0 ghz processor cluster ( ) , approximately five cpu months of calculations were required to fill t - db .
all data has been placed into a dedicated mysql server , with dual 2.8 ghz processors and 8 gb ram , running on fedora core 5  64 .
t - db has a query page with search parameters by pdb - code and chain identifier or by scop / astral ( 25 ) domain definitions . the results ( figure 1 ) of a search
provide a list of structurally similar proteins initially sorted by decreasing ne , which can be further restricted by number of alignments , by lower limit of z - score , by upper limit of the output rmsd and by length difference between query and subject .
all data is displayed inside a web - based hit - table with each structural neighbor in a single row .
for each hit the table shows the hit number , pdb - codes of query and subject proteins along with lengths , parameters of the structural alignment ( ne , rmsd , z - score , % sequence identity and positives ) , and the name of the subject protein .
finally , in an attempt to assist a user , vocabulary on the search and results pages has been linked to help content , which defines the terms to help aid in the analysis of the data .
after a search has been completed , there are several actions a user can conduct with the list of pre - computed structural neighbors ( figure 1 ) .
all individual alignments ( hits ) can be visualized over the web by using the 
align button in each row , resulting in the construction of an alignment plot , colored sequence alignment , and initialization of the friend applet ( 
the results of the search can be re - sorted by ne , rmsd , and z - score and hits can be removed by thresholds of ne , rmsd , z - score , and % identity .
results from the table can be saved as a comma delimited text format for spreadsheet analysis .
a set of every numeric parameter in the table can be displayed in a graph , with the selected value in sorted order .
the alignment is represented in three ways : as a graph , as a text alignment with residues colored by biochemical properties and as a file in fasta or sky ( friend specific ) formats . along with sequence data , files in sky format
contain reference to pdb - chains corresponding to sequences and a scripting section describing structure manipulation .
the locally installed version of friend executes the script right after all sequence and corresponding structures are loaded into memory . as a result one gets the multiple structural superimpositions corresponding to the alignment with differentiated aligned and unaligned residues automatically displayed in the friend software .
the aligned areas indicate the invariant regions which can be visualized to facilitate in studies of regions which contribute to structure stability along with functionally important active / ligand binding site residues .
t - server is a web - based server allowing users to submit their structures ( private or selected pieces , i.e. domains ) to be compared to all currently available structures in the pdb .
the user 's structures are uploaded by browser , or selected from the pdb , and a link is provided to the user to check whether calculations have finished ; optionally , the user can be notified by email about the calculation progress . upon completing the one - to - all calculation the results can be downloaded from the t - server web - page .
t - pair pairwise comparison server allows a user to align two structures , either identified by pdb - code and chain or uploaded , using the topofit method .
align button , additionally a summary email can be sent to a provided email address .
a large scale protein comparison was conducted which resulted in an improved z - score value .
the new z - score was derived based on a more accurate description of the produced random distribution of ne and rmsd .
the same dataset of non - related proteins used in the topofit paper were used in this new analysis .
the structures in the set were compared to produce the distribution of ne and rmsd representing the random model .
the distribution of ne for each value of rmsd was approximated by gaussian distribution with mean  = ne ( rmsd ) and  = ne ( rmsd ) depending on rmsd .
the parameters  and  were obtained from the least - squares fit of the experimental distributions for each value of rmsd . for a given rmsd and
ne the z - score was calculated as the deviation of ne from the gaussian average  normalized to the gaussian standard deviation . the dependences :  ,  +  ,  + 2 ,  + 3 ,  + 4 ,  + 5 were approximated by quadratic exponents ( figure 2 ) rather than by linear exponent as it was done in the original paper .
such an approximation allows for the calculation of z - score analytically instead of tabulating values of  and  for every value of rmsd . 

quadratic exponent fit ( solid line ) of dependencies  ,  +  ,  + 2 ,  + 3 ,  + 4 ,  + 5 ( dashed line ) .
the comparison ( data not shown ) between the new and old way of z - score estimation concluded that z - score values are significantly different for rmsd < 0.5  and > 1.5  , with new values being lower .
this resulted in the assignment of a lower significance to protein alignments sharing similarities in only short secondary structure elements .
currently t - db is useful for quick and effortless retrieval of statistically significant structural neighbors , along with automated viewing of t - db 's stored structural alignments in the friend software package for complete , thorough analysis .
additionally , a user can use t - server for the comparison of a structure not found in t - db , against the entire pdb , or use t - pair to align two structures ; providing functional utilities for the study of recently determined protein structures of unknown function .
the pipeline for updating t - db with topofit alignments for new structures has been developed , and new structures from the pdb will be compared and added once the calculations are completed .
future improvement will include : a deeper analysis between protein clusters , examination of the invariant cores in protein families , analysis of the variable regions across protein super - families , analysis of functional variations inside each cluster , downloadable similarity matrices within clusters , and graphically visualizing the distance between closely related clusters , allowing the users to browse from one cluster to neighboring clusters .
topofit 's non - parametric and objective way to separate the common part from the variable part , along with t - db 's accessibility will permit for these detailed studies to be shared with the scientific community .
 topofit - db ( t - db ) is a public web - based database of protein structural alignments based on the topofit method , providing a comprehensive resource for comparative analysis of protein structure families . 
 the topofit method is based on the discovery of a saturation point on the alignment curve ( topomax point ) which presents an ability to objectively identify a border between common and variable parts in a protein structural family , providing additional insight into protein comparison and functional annotation . 
 topofit also effectively detects non - sequential relations between protein structures . 
 t - db provides users with the convenient ability to retrieve and analyze structural neighbors for a protein ; do one - to - all calculation of a user provided structure against the entire current pdb release with t - server , and pair - wise comparison using the topofit method through the t - pair web page . 
 all outputs are reported in various web - based tables and graphics , with automated viewing of the structure - sequence alignments in the friend software package for complete , detailed analysis . 
 t - db presents researchers with the opportunity for comprehensive studies of the variability in proteins and is publicly available at . 
INTRODUCTION
BACKGROUND OF THE DATABASE
DATABASE CONTENT
QUERYING THE DATABASE
T-SERVER AND T-PAIR
None
SUMMARY AND FUTURE WORK