2.1. Known Small 3D-Patterns
2.1.1. Putative New Zinc Ion Coordination Sites
2.1.2. Promiscuous Binding Sites
2.1.3. Not Found Patterns
2.2. Serotonin Target Proteins
2.3. Finding/Discovering Unknown 3D-Patterns on Homology Model Structures
2.4. Comparison with other Methods for the Search and Description of Amino Acid Patterns
3. Materials and Methods
- Spacing Threshold (): This value is used to create the Virtual Grid of Coordinates and defines, how broad and rigorous will be the exploration of 3D-patterns. For instance, a = 0.5, means that every 0.5 Å in the 3D space of each protein structure, a new virtual coordinate of reference will be created. In all cases analysed in this work (Zinc finger C3H1-type containing proteins, serotonin target proteins and structures obtained from homology models), values were 0.8, 2 and 0.8 Å, respectively.
- Radius Threshold (): This term represents the limits of the size of the 3D-patterns searched. Low values are used to detect small binding sites ( e.g., 3 Å), whereas high values allow identification of bigger sites (e.g., 7 Å). In the two cases analyzed in this work (Zinc finger C3H1-type containing proteins, serotonin target proteins and structures obtained from homology models), values were 3, 5 and 2 Å, respectively.
- Displacement Threshold (): This value is used to expand the size and shape for the exploration of the 3D-patterns. By default, this value is set in 0, which means that only the spherical 3D-patterns are searched. If the user changes this value; for example, = 2, two new virtual centers will be considered for the searching of 3D-patterns. This option allows the obtaining of seven new elliptical/oval zones that will be explored to detect non spherical 3D-patterns (Supplementary Data, Figure S3).
- RMSD Threshold (): This value is used for clustering the 3D-patterns detected and represents a measure of structural variability for the sites composing each 3D-pattern. As mentioned in the Results, this parameter allows the comparison of a 3D-pattern with those, containing the same components (i.e., amino acid residues), previously found by 3D-PP. Thus, if the new site exceeds the threshold values defined by the user () when comparing it with the previously found site, a new cluster of the same 3D-pattern is created. Otherwise, the new 3D-pattern is included in the same cluster as that previously found. Therefore, this parameter is crucial for 3D-PP accuracy since it allows discrimination between 3D-patterns that contain similar components but exhibit a different topological conformations (i.e., amino acid residues which are not in the same spatial localization/order).
- Minimum Coverage (): This value allows the showing of only the 3D-patterns with a coverage value equal to or higher than .
3.1. Grid of Virtual Coordinates
- the min and max values of the X, Y, and Z axis of each structure are obtained.
- a virtual box whose size is determined by the previous values is defined (Figure 7A).
- the virtual box is filled by reference coordinates (x, y, z) distanced between them by an user-defined value (e.g., = 2 Å, Figure 7B).
- only the reference coordinates that show at least four residues surrounding (at a user-defined distance, e.g., = 5 Å) will be considered for the final grid (Figure 7C.)
3.2. Protein Preprocessing
- residues of the proteins surrounding each coordinate of the until a user-defined distance () are grouped.
- groups of residues with at least four components are considered as a site.
- a vector with the list of residues is defined for each site. Then, the sites are transformed into a representation of components through a sorted alphabetical list which contains the one letter code of the amino acid and the amount of occurrences of the same amino acid (e.g., the site “H31:I32:K10:K90:L11:L12:L7:P92:S3:S8:T9” is transformed into “1H1I2K3L1P2S1T”).
- If two different sites match in their representation of components (e.g., “1H1I2K3L1P2S1T”), the RMSD between these two sites is measured. If the RMSD exceeds the threshold values defined by the user (), a new cluster of the same 3D-pattern is created. On the contrary, a new site is added to the current cluster. This step is implemented to avoid two sites having the same components but different 3D conformations, being grouped in the same cluster. It should be noted that if the user set too permissive values (high values), there are more possibilities for grouping sites with different structural topologies; thus, many false positives can occur.
3.3. Creation of the Graph Databases
3.4. Result Visualization
- : The number of proteins in which a specific 3D-pattern was detected.
- : The number of proteins in which a specific 3D-pattern was not detected.
- : Level of conservation of a 3D-pattern in the set of proteins evaluated. The is calculated as follow:A high PCv value (e.g., 80%) indicates that a pattern containing a certain type of residues is found in many proteins (e.g., 80% of the proteins analyzed). It is worth noting that the sites composing a 3D pattern found do not necessarily exhibit the same structural topology in all the proteins in which such a pattern occurs.
- : Amount of sites (arrangement of residues) which are part of a specific 3D-pattern.
- : This value represents the structural variability of a 3D-pattern. Thus, a low number of clusters denotes low variability and, on the contrary, a high number of clusters is indicative of several structural conformations (with different topologies) of sites forming a 3D-pattern.
- : Represents the cluster with the highest coverage on each 3D-pattern.
- : Amount of sites (arrangement of residues) which are part of a specific cluster.
- : The number of proteins that contain a particular 3D-pattern.
- : Level of conservation of a cluster in the set of proteins belonging to a particular 3D-pattern. The is calculated as follows:A high CCv value (e.g., 80%) indicates that a pattern with the same structural topology is present in most of the proteins (80%) of the corresponding cluster.
- : This button shows a multiple sequences-based alignment of the residues of each site of a specific cluster.
- : This button displays a jsmol viewer with multiple structural-based alignments of the residues of each site of a particular cluster.
- : Information of the name and number of the residues forming the site.
- : Name of the protein where the site was detected. This variable can be the PDBid or the name of the file, in the case of homology models.
- : The chain where the site was detected.
- : Root mean square deviation of atomic positions of the particular site against the reference site.
- : Referential coordinate of the from where a specific site was detected.
- : This button shows a jsmol viewer loading the protein and highlighting the residues corresponding to a particular site.
Conflicts of Interest
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|Item||PROSITE (A)||3D-PP (B)||PDBsum(C)||A & B||A & C||B & C||A & B & C|
|amount of sites||125||152||124||123||124||122||122|
|Tool||X-ray/Homology Model||Require Known Pattern/Ligand||Comments|
|Catalytic site identification—a web server to identify catalytic site structural matches throughout PDB ||Yes/No||Yes||The most similar section to replicate our experiments with this tool is “Find CSA catalytic sites in your proteins.” However, it was not possible to obtain the results because only one PDB structure can be processed at the same time. When we tried to upload a homology model the server returned a “Server Error (500)”.|
|MMDB and VAST+ ||Yes/No||Yes||This tool allows the finding of 3D-patterns of amino acids in only one PDB structure at the same time. It is not possible to identify conserved 3D-patterns on a set of protein structures.|
|IMAAAGINE ||Yes/No||Yes||This tool allows searching 3D-patterns of amino acids in the entire PDB database. The user must define a structural description of query. It is not possible to identify conserved 3D-patterns on a set of protein structures.|
|PatternQuery ||Yes/No||Yes||This tool allows for the searching of 3D-patterns of amino acids in the PDB database or in a particular data set of protein structures. The user must define a detailed structural description of the query (known 3D-pattern). It is not possible to identify conserved 3D-patterns on a set of protein structures.|
|ProBiS ||Yes/Yes||No||This tool search all the 3D-patterns of amino acids associated to a ligand or functional annotations, present in a queried protein structure. Then these 3D-patterns are searched on the entire PDB database. It is not possible to identify conserved 3D-patterns on a set of protein structures.|
|Geomfinder ||Yes/Yes||No||This tool compares all the possible 3D-patterns of amino acids of one protein structure with all the possible 3D-patterns of a second protein structure. The identification of the 3D-patterns works separately on each pair of protein structures, and the results are not matched. Therefore, it is not possible to identify conserved 3D-patterns in a set of protein structures.|
|MultiBind ||Yes/Yes||No||This tool identifies similar 3D-patterns among a list of PDBids. With this tool, we could find conserved 3D-patterns, but the server did not work with our data sets. In the case of the homology models, the measures can not be assessed because our structures do not contains ligands. The server returns the following comment “No valid ligand. Can not define the query binding site”.|
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