For aspartate, glutamate, arginine, and lysine, the charged state is the default entry, whereas His is neutral. Please, spend time to optimize the hydrogen bond pattern for your target. This is especially true for deeply buried residues, for which the charged state seems less favored, and polar interactions to ligand atoms are less expected.

For histidine, it is not primarily the protonation itself but the tautomers to be differentiated: The ring system can be protonated at two different N atoms; one carries the PDB notation ND2, the other one NE1. Alternatively, both nitrogen atoms may be protonated. We recommend to carefully inspect the orientation of adjacent hydrogen-bond donors and -acceptors (if available in the context of the ligand, cofactors and metal ions): Are there interactions involved to any of these histidine isomers? Which is the more reasonable protomer? Finally, cysteine residues are often involved in forming disulfide bridges: It is of crucial importance to tell FlexX that the respective Cys atoms do not carry hydrogen atoms: please assign the proper template as it exists in the rdf file.

A template code consists of the amino acid three letter code plus - depending on the situation - some modifiers:

h		cysteine not involved in s-s bond
1/2		His tautomer assignment
+/-		charge for ionic side chain
n(+)		N-terminal, possibly charged
c(-)		C-terminal, possibly charged

So, the +/- selects the charged variants, cysh would correspond to a single, protonated cysteine, and cys would be defined to act as a disulfide-bridge former. Histidine may appear as his1/his2/his+ and so on. A complete list of all possible templates can be found in the static data file amino.dat. It is also possible to extend amino.dat with user defined templates, e.g. if non-standard amino acids occur in the pdf file.

Here is an example:

@templates
ARG * * arg+
ASP * * asp-		# aspartate, charged by default
CYS * * cysh
GLU * * glu-
ASP A 10 asp		# keep aspartate 10 in chain A neutral
...

N- and C-termini are special residues, and the templates for them are assigned to any amino acid that occurs at the end of a peptide chain. Here of course, the protonation is controlled by the +/- extension of nterm and cterm.

* * first nterm+
* * last cterm-

Here is an example of usage of the above-mentioned modifiers:

* * first ala+n+

with the effect to protonate the first alanine which occurs in the respective chain.

Advanced Use To Avoid a Known FlexX Enemy: Charged Amino Acids at the Rim of Active Sites
It has been encountered a couple of times that charged amino acids at the edge of a pocket act as "magnets" pulling the ligands out of the pockets when docking. You can usually avoid this quite well by checking for their existence and assigning neutral templates to them.

The "correct" hydrogen positions have to be defined in the @h_torsions section of the RDF file. The first entry contains the template name. The next two entries allow for restricting the definition to particular amino acids by providing the chain_id and the number of the amino acid. Wildcards are allowed (and often used, see example below) for the first three parameters. The position of the hydrogen is defined by the last four parameters providing (in this sequence) the first, second, and third atom with the implicit hydrogen as fourth atom as a reference for the rotatable bond between the second and third atom. The last parameter is the torsion angle for this bond. See below as an example the default entries for serine, threonine, tyrosine, and protonated cysteine:

@h_torsions
cysh*   *       *       _ca      _cb      _sg      180.
glu     *       *       _oe2     _cd      _oe1     0.
ser*    *       *       _ca      _cb      _og      180.
tyr*    *       *       _ce1     _cz      _oh      180.
thr*    *       *       _ca      _cb      _og1     180.

Type 'SELINIT' and then the number of the level you would like to turn on, or 'SELINIT !' and the number of the level you would like to turn off. E.g. 'SELINIT 4' would turn on, 'SELINIT !4' would turn off the protonation.