the human genome appears to encode no more than 25  000 proteins ( 1 ) . this relatively small number of genes compared with the genome of other species has been one of the big surprises to come out of the human genome project . a major challenge is to understand how post - translational events affect the activities and functions of these proteins in relation to health and disease . among these , glycosylation is by far the most frequent ; more than half of all the proteins in the human body have glycan molecules attached ( 2,3 ) . their oligosaccharide moieties are implicated in a wide range of cell  cell and cell  matrix recognition events . therefore , only a small number of glycan structures are available for which sufficient electron density for an entire oligosaccharide chain can be detected ( 4 ) . unambiguous structure determination based on nmr - derived geometric constraints alone is often not possible ( 5 ) . time - consuming computational approaches such as monte carlo calculations and molecular dynamics simulations have been widely used to explore the conformational space accessible to complex carbohydrates ( 6,7 ) . for reasons that are not well understood , unfortunately , the unambiguous determination of occupied n - glycosylation sites is experimentally demanding and can vary between different cellular locations . the aims of glyprot are ( i ) to evaluate whether a potential n - glycosylation site is spatially accessible , ( ii ) to generate reasonable three - dimensional ( 3d ) models of glycoproteins with user - definable glycan moieties and ( iii ) to provide some evidence on how the physicochemical parameters can change between the varying glycoforms of a protein . the 3d structure of a protein in protein data bank ( pdb ) format is required as input ( see dataflow given in figure 1 ) . the protein structure can be either taken directly from the pdb or uploaded from a local computer . potential n - glycosylation sites ( sequon : asn - x - ser / thr , where x is not pro ) are automatically detected and highlighted using the one - letter amino acid code . in cases where experimental coordinates with already attached glycans are provided , the internal coordinates ( distance between the n of the asn - sidechain and the c1 of the attached -d - glcpnac and the torsion angles determining the orientation of the glycan moiety ) are displayed . the orientation of the attached n - glycan relative to the glycosylation site is described by the four consecutive torsion angles 1 , 2 ,  and  ( for definition see table 1 ) . it is well known from the analysis of the experimentally available 3d structures of glycoproteins ( 8,9 ) that preferred orientations of the glycan moiety relative to the protein exist ( figure 2 ) . conformational maps indicating the populated areas for all four torsion angles can be easily obtained using the glytorsion tool ( ) from the carbohydrate structure suite ( 10 ) . it is assumed that the man3 n - glycan core exhibits one dominant , relatively rigid conformation . this assumption is supported by the analysis of experimentally determined torsion angles for the corresponding glycosidic linkages in the pdb ( table 2 and figure 3 ) . only the 16 linkage exhibits two significantly populated conformations , whereas the other three linkages constitute only one highly populated conformation . to evaluate whether a potential glycosylation site is spatially accessible , a program written in c is used to connect the man3 n - glycan core to the protein and test all possible angle sets . the frequency of occurrence of the four relevant torsion angles ( table 1 ) is used to orient the n - glycan core . next , the program evaluates whether atoms of the attached glycan moiety overlap with the protein . if spatial overlaps are detected , the model is rejected and the next most frequently observed orientation of the glycan moiety is applied . table 1 lists the values of the four relevant torsion angles and the succession in which they are applied . this procedure is repeated until a structure with no or minor overlap has been found . if all orientations listed in table 1 have been applied and all resulting glycoprotein structures exhibit overlapping atoms , it is assumed that the glycosylation site is spatially inaccessible and therefore can not be glycosylated . for each spatially accessible potential n - glycosylation site three the user can 
 select the type of n - glycan ( e.g. oligomannose rich , complex , hybrid , very large ) ; by default a typical structure for each class is taken;select an n - glycan from a database of > 1000 structures ( figure 4 ) constructed using sweet - ii ( 11 ) and optimized using the tinker mm3 force field ( ) ; the database is searchable by n - glycan composition;construct the desired n - glycan using sweet - ii by user input of the desired structure using the extended iupac nomenclature . 
 if the coordinates provided already contain attached n - glycans , the user can either accept this orientation or use the procedure described above to align the glycan moiety . select the type of n - glycan ( e.g. oligomannose rich , complex , hybrid , very large ) ; by default a typical structure for each class is taken ; select an n - glycan from a database of > 1000 structures ( figure 4 ) constructed using sweet - ii ( 11 ) and optimized using the tinker mm3 force field ( ) ; the database is searchable by n - glycan composition ; construct the desired n - glycan using sweet - ii by user input of the desired structure using the extended iupac nomenclature . the orientation of the attached n - glycan relative to the glycosylation site is described by the four consecutive torsion angles 1 , 2 ,  and  ( for definition see table 1 ) . it is well known from the analysis of the experimentally available 3d structures of glycoproteins ( 8,9 ) that preferred orientations of the glycan moiety relative to the protein exist ( figure 2 ) . conformational maps indicating the populated areas for all four torsion angles can be easily obtained using the glytorsion tool ( ) from the carbohydrate structure suite ( 10 ) . it is assumed that the man3 n - glycan core exhibits one dominant , relatively rigid conformation . this assumption is supported by the analysis of experimentally determined torsion angles for the corresponding glycosidic linkages in the pdb ( table 2 and figure 3 ) . only the 16 linkage exhibits two significantly populated conformations , whereas the other three linkages constitute only one highly populated conformation . to evaluate whether a potential glycosylation site is spatially accessible , a program written in c is used to connect the man3 n - glycan core to the protein and test all possible angle sets . the frequency of occurrence of the four relevant torsion angles ( table 1 ) is used to orient the n - glycan core . next , the program evaluates whether atoms of the attached glycan moiety overlap with the protein . if spatial overlaps are detected , the model is rejected and the next most frequently observed orientation of the glycan moiety is applied . table 1 lists the values of the four relevant torsion angles and the succession in which they are applied . this procedure is repeated until a structure with no or minor overlap has been found . if all orientations listed in table 1 have been applied and all resulting glycoprotein structures exhibit overlapping atoms , it is assumed that the glycosylation site is spatially inaccessible and therefore can not be glycosylated . for each spatially accessible potential n - glycosylation site three options are offered for selecting the n - glycan to be connected . the user can 
 select the type of n - glycan ( e.g. oligomannose rich , complex , hybrid , very large ) ; by default a typical structure for each class is taken;select an n - glycan from a database of > 1000 structures ( figure 4 ) constructed using sweet - ii ( 11 ) and optimized using the tinker mm3 force field ( ) ; the database is searchable by n - glycan composition;construct the desired n - glycan using sweet - ii by user input of the desired structure using the extended iupac nomenclature . if the coordinates provided already contain attached n - glycans , the user can either accept this orientation or use the procedure described above to align the glycan moiety . select the type of n - glycan ( e.g. oligomannose rich , complex , hybrid , very large ) ; by default a typical structure for each class is taken ; select an n - glycan from a database of > 1000 structures ( figure 4 ) constructed using sweet - ii ( 11 ) and optimized using the tinker mm3 force field ( ) ; the database is searchable by n - glycan composition ; construct the desired n - glycan using sweet - ii by user input of the desired structure using the extended iupac nomenclature . the atomic coordinates of the desired glycoprotein are given in pdb format , and they are immediately displayed using the java applet jmol ( ) . the coordinates can be downloaded and used as input for many 3d visualization programs ( see figure 5 ) . in addition , some physicochemical parameters for the non - glycosylated and the glycosylated protein are displayed to provide a general delineation of the changes caused by the selected glycoform ( see table 3 ) . the program surface racer ( 12 ) the generally observed increase of the polar surface area as a result of glycosylation reflects the well - known experience that glycoproteins exhibit higher solubility . although it is estimated that > 50% of all proteins are glycosylated ( 2,3 ) , only 5% of all pdb entries have attached glycan chains ( 4 ) . moreover , only a few entries in the pdb contain x - ray diffraction data with sufficient electron density to detect an entire oligosaccharide chain . the 3d models of glycoproteins constructed with glyprot can provide some evidence on which areas of a protein are captured by a certain glycoform and whether , for example , a binding site is covered so that the biological activity of a protein may be influenced . simply because of their large size and hydrophilicity , glycans can alter the physicochemical properties of a glycoprotein , making it more soluble , reducing backbone flexibility and thus leading to increased protein stability , protecting it from proteolysis , and so on . the calculation of some characteristic physicochemical parameters will help in the evaluation and explanation of the varying properties of different glycoforms . of the therapeutic proteins on the market , 60% are glycoproteins ( 13 ) . often , the removal of n - glycans results in a protein with a very short half - life and virtually no activity in vivo ( 13 ) . a comprehensive evaluation of the impact of varying glycoforms on protein function is hampered by the high conformational flexibility of glycan structures . based on the statistical analysis of experimentally known glycan conformations , glyprot constructs a reasonable conformation out of a manifold . however , a more realistic analysis would require the complete conformational space that is accessible to a glycan at a given glycosylation site to be sacnned . therefore , we intend to expand the glyprot service with an option allowing the exploration of the conformational space accessible to an n - glycan , which is covalently bound to a specific glycosylation site . a similar approach has already been successfully applied to rapidly generate a representative ensemble of conformations of single n - glycan molecules ( 6 ) . this algorithm is based on a comprehensive set of conformations of n - glycan fragments that were derived from molecular dynamics simulations . however , this approach would assume a protein conformation that remains unchanged through the attachment of varying glycans . in order to allow conformational changes of the protein backbone , only force - field - based , time - consuming simulation approaches such as molecular dynamics with inclusion of explicit water molecules would be appropriate . statistical analysis of the pdb for torsion angles determining the orientation of the glycan moiety relative to the protein . statistical analysis of the pdb for glycosidic torsion angles determining the conformation of the n - glycan core . input spreadsheet ( top ) used to query the database , which contains > 1000 3d structures of n - glycans ( bottom ) . the protein part is given as a cartoon representation ; the glycan part as a spacefill model . definition of torsion angles defining the orientation of the glycan moiety relative to the protein and hierarchy of applied torsion angles torsion angles for glycosidic linkages of the n - glycan core region analysis of the pdb entries was carried out with the glytorsion tool . with  = oring  xa  o  x and  = xa  o  x  cg1 . comparison of some characteristic physicochemical properties of the pure influenza a subtype n9 neuraminidase ( 14 ) and the constructed glycoform asa , solvent accessible surface area ( in  ) .
glyprot ( ) is a web - based tool that enables meaningful n - glycan conformations to be attached to all the spatially accessible potential n - glycosylation sites of a known three - dimensional ( 3d ) protein structure . 
 the probabilities of physicochemical properties such as mass , accessible surface and radius of gyration are calculated . 
 the purpose of this service is to provide rapid access to reliable 3d models of glycoproteins , which can subsequently be refined by using more elaborate simulations and validated by comparing the generated models with experimental data .