1. Specify the input sequences

All the input sequences must be in one-letter amino acid code. The allowed alphabet (not case sensitive) is as follows:

A C D E F G H I K L M N P Q R S T V W Y and X (unknown)

The characters not in the one-letter amino acid code e.g. letters like 'B' or 'Z', digits and other non-alphabetic symbols will all be converted to X before processing; they will be accepted but not used when making the predictions. White space within the sequences, if any, will be ignored. The sequences can be input in the following two ways:

• Paste a single sequence or several sequences in FASTA format into the input field.
  Note: if you paste more than one sequence FASTA format must be used.

• Submit a file in FASTA format directly from your local disk. The file may contain multiple sequences.

Both methods may be used in the same submission. Peptides shorter than 9 residues can produce non-reliable results. Always include 4 residues on both sides of the Tyr/Ser/Thr you want evaluated. The optimal is to use the complete "native" protein sequence.

2. Customize your run

By default the server predicts phosphorylation sites for all the serine, threonine and tyrosine residues in the input sequences, displays all the predictions made for each such residue and generates graphical output illustrating the results. These settings can be changed, as follows:

• "Residues to predict" - the scope of prediction can be limited to just one of serine/threonine/tyrosine. The deafult is to predict for all the three amino acids.

• "For each residue display only the best prediction" - for each residue the server can be made to display only the prediction with the highest score. The default is to display all the predictions for all the residues.

• "Display only the scores higher than ..." - only the predictions with scores higher than a given threshold will be displayed. By default all the predictions are displayed (the threshold of "0"). NOTE: the choice of "0.5" will imply that only the positive predictions will be shown (see the Output format).

• "Output format" - by default the output format is the native NetPhos format (see the Output format). It can be changed to GFF.

• "Generate graphics" - by default the server produces graphical output illustrating the predictions. This can be disabled.

Before sumitting larger runs, the user is advised to test the settings above on a few sequences, to arrive at the best combination for a given run.

3. Submit the job

When ready press the button labelled 'Submit'. The status of your job (either 'queued' or 'running') will be displayed and constantly updated until it terminates and the server output appears in the browser window.

At any time during the wait you may enter your e-mail address and simply leave the window. Your job will continue; you will be notified by e-mail when it has terminated. The e-mail message will contain the URL under which the results are stored; they will remain on the server for 24 hours for you to collect them.