MolDraw › Chemistry tools › Protonation States at pH
Add Hydrogens at pH: Protonation States
In short: Pick a pH. For a protein, get the state of every His, Asp, Glu, Lys, Arg, Cys, Tyr and terminus with AMBER names (HID/HIE/HIP, ASH, GLH, LYN, CYX). For a ligand, get the charged form as SMILES and SDF. It is free and runs in your browser.
PDB with protonation-state residue names
No hydrogen coordinates are written for proteins: placing rotatable OH, NH3⁺ and SH hydrogens correctly needs an optimiser. Load this PDB into AMBER tleap or pdb4amber, which build hydrogens from the residue names, or into PDB2PQR / Reduce.
Charged form (SMILES)
SDF with all hydrogens (2D coordinates)
The SDF has flat 2D coordinates. Generate 3D coordinates (for example with the SMILES to 3D converter, RDKit or Open Babel) before docking.
Model pKa values used for proteins
A group is set to its protonated form when pH < pKa. The fraction protonated is 1 / (1 + 10pH − pKa). Rows within one pH unit of the pKa are highlighted: both forms are present in real samples.
| Group | pKa | Below pKa | Above pKa | At pH 7.4 |
|---|---|---|---|---|
| C-terminus (COOH) | 3.67 | COOH, 0 | COO⁻, −1 | COO⁻ |
| Asp | 3.67 | ASH, 0 | ASP, −1 | ASP |
| Glu | 4.25 | GLH, 0 | GLU, −1 | GLU |
| His | 6.54 | HIP, +1 | HIE or HID, 0 | HIE (12% HIP) |
| N-terminus (NH₂) | 8.00 | NH₃⁺, +1 | NH₂, 0 | NH₃⁺ (80%) |
| Cys | 8.55 | CYS, 0 | CYM, −1 | CYS |
| Tyr | 9.84 | TYR, 0 | phenolate, −1 | TYR |
| Lys | 10.40 | LYS, +1 | LYN, 0 | LYS |
| Arg | 12.3 | ARG, +1 | neutral | ARG |
Values for alanine pentapeptides from Thurlkill, Grimsley, Scholtz & Pace, Protein Science 2006, 15, 1214. Arg from Nozaki & Tanford (1967); later work puts it near 13.8, which does not change its state.
Rules that do not depend on pH
- Disulfides: two Cys with SG–SG under 2.5 Å become CYX.
- Metal-bound Cys: SG within 2.8 Å of Zn, Fe, Cu, Ni, Co, Mn, Cd or Hg becomes CYM (thiolate).
- Metal-bound His: the ring N that binds the metal carries no H, so the other tautomer is chosen (ND1 bound → HIE, NE2 bound → HID).
- Other neutral His: HIE by default. Choosing HID or HIE correctly needs a hydrogen-bond analysis, which this tool does not do.
Small-molecule rules
The molecule is first neutralised, then each recognised group is ionised if the pH is on the far side of its typical pKa. These are group averages; substituents can move a real pKa by 1–3 units.
| Group | Typical pKa | At pH 7.4 |
|---|---|---|
| Sulfonic acid, sulfate ester | ≈ −1 | anion |
| Phosphate / phosphonic acid, 1st OH | ≈ 2 | anion |
| Phosphate monoester, 2nd OH (phosphonic 7.5, phosphoric 7.2) | ≈ 6.5 | dianion (near pKa) |
| Carboxylic acid (aliphatic / aromatic) | 4.8 / 4.2 | carboxylate |
| Tetrazole, acyl sulfonamide | 4.5–4.9 | anion |
| Thiophenol / aliphatic thiol | 6.6 / 10 | anion / neutral |
| Hydroxamic acid | ≈ 9 | neutral |
| Phenol | ≈ 10 | neutral |
| Primary / secondary / tertiary aliphatic amine (−1 if benzylic) | 10.6 / 11.0 / 9.8 | cation |
| Second N of a 1,2-diamine (piperazine, ethylenediamine) | ≈ 6 | neutral |
| Amidine / guanidine (acylguanidine 8) | 12.4 / 13.6 | cation |
| Imidazole (benzimidazole 5.5) | 7.0 | neutral (near pKa) |
| Pyridine / aniline | 5.2 / 4.6 | neutral |
How to use it
- Choose Protein (PDB) or Small molecule.
- Set the pH: 7.4 for blood plasma, about 5 for lysosomes or an acidic assay buffer.
- Load the structure: a cleaned PDB (see the PDB cleaner), a PDB ID, a SMILES string or a MOL/SDF block.
- Check highlighted rows. They are near their pKa and are worth a second look with PROPKA.
- Download the renamed PDB for tleap or PDB2PQR, or the charged ligand SDF. Then convert to PDBQT.
Limitations
- One pKa per residue type. No desolvation, hydrogen-bond or charge–charge shifts. Active-site Asp, Glu, His, Cys and Lys are the residues most likely to be wrong.
- No hydrogen coordinates for proteins, and no Asn/Gln/His side-chain flips.
- Chain ends next to missing residues are treated as real termini, as most preparation tools do.
- Small molecules: groups not in the table (sulfonamides, imides, N-heteroaryl sulfonamides, enols, aminopyridines, amines next to CF₃ or carbonyls) are left as drawn or use a default value that may be off by several units. Tautomers are not enumerated.
- Only the first record of a multi-molecule SDF is read, and the SDF output has 2D coordinates.
- For drug-like ligands, Epik, Dimorphite-DL, OpenEye or ChemAxon give per-site pKa predictions.
Frequently asked questions
Is histidine protonated at pH 7.4?
Mostly not. With the model pKa of 6.54, about 12% of free histidine is protonated at pH 7.4, so it is set to a neutral tautomer (HIE by default). At pH 5 it is about 97% protonated and is set to HIP. In a real protein the pKa can shift by several units.
What is the difference between HID, HIE and HIP?
They are AMBER names for histidine. HID has a hydrogen on ND1 (delta nitrogen), HIE on NE2 (epsilon nitrogen); both are neutral. HIP has hydrogens on both and carries +1. CHARMM calls them HSD, HSE and HSP.
Is this the same as PROPKA or PDB2PQR?
No. This tool uses fixed model pKa values for each residue type. PROPKA and PDB2PQR estimate a pKa for each residue from its surroundings, such as burial, hydrogen bonds and nearby charges. Use them for publication work, enzyme active sites and buried residues.
Does the tool add hydrogen atoms to the protein?
No. It writes the protonation state into the residue names (for example HIP, ASH, LYN, CYX). AMBER tleap, pdb4amber, PDB2PQR or Reduce then build hydrogens with correct geometry. For small molecules the SDF output does include all hydrogens, with 2D coordinates.
Why is acetic acid charged at pH 7.4?
Its pKa is about 4.8, so at pH 7.4 the ratio of acetate to acetic acid is 10^(7.4 − 4.8), about 400 to 1. The tool outputs acetate, CC([O-])=O. Phenol (pKa about 10) stays neutral and ethylamine (pKa about 10.6) becomes ethylammonium.
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