Handling hydrogen atoms

Servalcat provides options for handling hydrogen atoms during refinement. Because hydrogen atoms are often difficult to observe at typical resolutions, their treatment involves geometric restraints and controlling whether they respond to the experimental data.

Note

By default, hydrogen atoms are used to maintain proper geometry and contribute to structure factor calculations, but their positions are only driven by geometric restraints. To allow their positions to be actively shifted by the experimental data, you must explicitly enable them.

Control keywords

The following options are available for refine_xtal_norefmac and refine_spa_norefmac.

--hydrogen <all|yes|no>

Specifies how hydrogen atoms are treated or generated before refinement. This option inherits the behaviour of the make hydr keyword in Refmac with slight differences.

• all (default): Hydrogen atoms are (re)generated for the model. However, “rotatable” hydrogen atoms (where multiple conformations are geometrically possible, such as hydroxyl hydrogens in Ser/Thr/Tyr) are not generated. This prevents incorrect initial placement from introducing artificial steric clashes.

  Note

  Histidine is a special case under all. While HD1 and HE2 are present in the standard definition, they are not generated automatically by all due to tautomeric ambiguity.

• yes: Hydrogen atoms are used exactly as they are present in the input model. No new hydrogens are generated, and existing ones are not removed.

• no: All hydrogen atoms are stripped from the model before refinement.

--refine_h

By default, hydrogen atoms contribute to geometric restraints and the calculation of structure factors, but they are excluded from the gradient calculation against the experimental data (i.e., their positions are not shifted by the data).

Including the --refine_h flag includes hydrogen atoms in the gradient calculation of the experimental data term, allowing their positions to be refined against the observations.

Note

Regardless of the settings, torsion angle restraints for hydrogen atoms are always active. These restraints are defined in the standard monomer library or user-supplied dictionary files.

--hout

Controls the output of hydrogen atoms. By default, hydrogen atoms are not written to the coordinate file. Specify --hout to include hydrogen atoms in the output PDB/mmCIF files.

Generating all hydrogen atoms explicitly

To generate all hydrogen atoms including those with multiple potential ideal positions prior to refinement, use the following utility command:

servalcat util h_add input.pdb

This command places all hydrogen atoms at one of their ideal positions. It does not account for potential steric clashes with neighbouring atoms. Users must inspect the resulting positions and adjust them manually if necessary before proceeding to refinement. For the subsequent refinement, use --hydrogen yes --hout.

Tautomers and protonation states

Servalcat follows the monomer library definitions for tautomers and protonation states. These definitions are generally intended to approximate standard protonation states under typical physiological conditions (e.g., carboxyl groups are deprotonated, and amino groups are protonated).

To use alternative tautomers or protonation states, use the modification mechanism. The following modifications are defined within the monomer library:

• ASPprot: Protonated aspartate

• GLUprot: Protonated glutamate

• HISprot1: Histidine tautomer with HE2 (HD1 absent)

• HISprot2: Histidine tautomer with HD1 (HE2 absent)

To apply these modifications to specific residues, use MODRES records (for PDB files) or the _pdbx_struct_mod_residue category (for mmCIF files).

We use the unused column (positions 73-80) in MODRES:

MODRES      GLU A    7                                                  GLUprot

and we use our own addition ccp4_mod_id in _pdbx_struct_mod_residue:

loop_
_pdbx_struct_mod_residue.id
_pdbx_struct_mod_residue.auth_asym_id
_pdbx_struct_mod_residue.auth_seq_id
_pdbx_struct_mod_residue.PDB_ins_code
_pdbx_struct_mod_residue.auth_comp_id
_pdbx_struct_mod_residue.label_comp_id
_pdbx_struct_mod_residue.parent_comp_id
_pdbx_struct_mod_residue.details
_pdbx_struct_mod_residue.ccp4_mod_id
1 A 7 ? GLU GLU ? ? 'GLUprot'