One of the following input options should be selected by marking the relevant radio button:
If the protein structure is available either (1) enter the PDB ID or (2) upload the PDB file. In any case enter the required chain identifiers. If the protein structure is unavailable either (3) enter the protein sequence or (4) upload the protein sequence file.
Each structure in the Protein Data Bank (PDB) is represented by a four character alphanumeric identifier, assigned upon its deposition. For example, 1a2y is the identification code for the hen egg white lysozyme in complex with mouse monoclonal antibody D1.3. If a user wishes Epitopia to download a specified PDB file, he should enter a valid PDB ID. The type of structure can either be an X-ray crystal structure, or a representative NMR model. For more information go to the PDB web-site.
Uploading a PDB file
An alternative way to provide a structure input is to upload a local PDB file. If this option is selected the PDB file must contain the relevant SEQRES and ATOM records. See the PDB file format for further information.
Selecting chain identifiers
The required chain identifiers should be defined by the user. More than one chain can be defined, and in that case the required chain identifiers should be concatenated without space separating them (e.g., 'LH' should be used for defining chains L and H). If the user wishes to select all the chains in the structure, the term "all" can be used instead of specifying all chain identifiers.
Keeping non-selected chains
If the selected input structure contains more chains than defined by the user, by default Epitopia disjoints the selected chains from the remaining structure, and the computation proceeds using the selected disjointed structure (i.e., removes the non-selected chains). If the user wishes to run Epitopia without disjointing the selected chains, the "Keep non-selected chains" checkbox should be marked. In that case, the structural considerations for computing the immunogenicity scores will be affected by all the chains in the structure, but the immunogenicity scores themselves will only be computed for the residues of the selected chains.
Entering a protein sequence
A protein sequence may be pasted to the relevant window. The sequence should contain only standard amino acids. The pasted sequence should be in Fasta format, where the header line is optional.
Uploading a protein sequence file
An alternative way to provide a sequence input is to upload a file containing the sequence. The sequence should contain only standard amino acids. The file should be in Fasta format, where the header line is optional.
Entering an e-mail address
Since an Epitopia run make take a while it is advisable to provided an e-mail address to which a link to the results page will be sent once Epitopia completes its computation.
Understanding the output for the structure option
For each solvent accessible residue of the selected chains in the input structure, Epitopia computes an immunogenicity score and a corresponding probability score. The output file lists these scores for each residue (the higher the scores are, the more immunogenic the residue is and more probable that it was drawn from a population of epitope residues, respectively) and a character indicating whether the residue is exposed to the solvent ("E"), or buried ("B"). The records are listed in descending order according to the immunogenicity scores.
The distribution of immunogenicity scores is divided to five equally-sized score categories. Each category is assigned a color according to the color-coding scheme. The FirstGlance in Jmol visualization tool displays the input structure, where all residues are displayed in spacefill mode. Residues of the selected chains are colored light gray, and residues of the non-selected chains are colored dark gray. The solvent exposed residues of the selected chains are colored according to their score category. The FirstGlance in Jmol visualization tool enables the user to further control the modes of representation.
An alternative to using the online FirstGlance in Jmol visualization is to use RasMol visualization tool offline. The script for coloring the PDB file according to Epitopia's prediction in RasMol is an additional output provided by Epitopia.
Understanding the output for the sequence option
For each residue in the input sequence, Epitopia computes an immunogenicity score and a corresponding probability score. The output file is similar to that of the structure option, except for the buried/exposed indicator which is based on prediction rather than direct computation.
The distribution of immunogenicity scores is divided to five equally-sized score categories. Each category is assigned a color according to the color-coding scheme. The sequence is displayed where each residue is color-coded according to its score category, and the buried/exposed indicator is also shown.
Epitopia applies a search procedure for clustering highly immunogenic residues in order to find candidate epitopes in a sequence input. The output of this procedure is a list of the five top-scoring stretches, where for each stretch its rank, the statistical statistical significance (p-value) of its score, the number of residues comprsing it, and the residues themselves are listed.
The immunogenicity scale: