The Protein-Ligand Interaction Profiler (PLIP) is a tool to analyze and visualize protein-ligand interactions in PDB files.
- Eight types of noncovalent interactions
- Interaction between proteins and
- small molecules
- ions
- polymers
- DNA / RNA
- Rich additiona information on binding, e.g. unpaired functional groups
- Direct download of PDB structures from PDB server
- Automatic detection and grouping of relevant ligands in a PDB file
- No need for special preparation of a PDB file, works out of the box
- Automatic fixiing of errors in PDB files
- Processing of custom PDB files containing protein-ligand complexes (e.g. from docking)
- Atom-level interaction reports in TXT and XML formats for easy parsing
- Generation of PyMOL session files (.pse), enabling easy preparation of images for publications and talks
- Rendering of 3D interaction diagram for each ligand and its interactions with the protein
The following instructions work on a unix machine. If you are using Windows or OSX, the process may differ. Please refer to the webpages of the corresponding tools to get help for installation.
To install all dependencies for PLIP, either just install PyMOL and then use the pip installation routine or install all tools and clone from GitHub.
The current version of PLIP depends on
- OpenBabel >=2.3.2
- PyMOL 1.7.x with Python bindings
- Imagemagick >=6.9.x (optional)
and should be executed with Python 2.7.x.
Example command for Ubuntu using apt-get
sudo apt-get install pymol openbabel python-openbabel imagemagickThere are several options to get PLIP on your machine.
If you have the Python package manager pip installed, you can install PLIP in a terminal via the following command:
pip install plipIf you want to clone from Github, open a new system terminal and clone the repository using
git clone https://github.com/ssalentin/plip.git ~/pliptoolYou can use any other user directory, but we will use the one given above for the documentation.
The package manager pip will create symlinks for you so you can call PLIP using plip in the command line. If you cloned from Github, use the following command to do the same:
alias plip='~/pliptool/plip/plipcmd'Having PLIP installed, you can run
plip -hto list all available command line options. We will go through all important settings in this section. A typical application of PLIP involves the analysis of protein-ligand interactions in a structure from the Protein Data Bank (PDB). PLIP can automatically fetch the entry from the PDB server when a valid PDB ID is provided.
plip -i 1vsn -vThe command above will fetch the PDB entry 1vsn from the server, analyze all interactions and print out the results (verbose mode). No output files are produced at this point.
The same can be done for local PDB files.
wget http://files.rcsb.org/download/1EVE.pdb
plip -f 1EVE.pdb -vThe output formats can be added in any combination, currently including:
- XML report files (
-x, best for automatic processing) - Text report files (
-t, human-readable) - PyMOL session files (
-y) - Ray-traced images (
-p)
plip -i 1osn -pyxThe command above will fetch the PDB entry 1osn, analyze all interactions and produce one XML result file (-x) as well as rendered images (-p) and PyMOL session files (-y) for each binding site.
PLIP can process multiple structures at once, either from PDB or local files. To activate batch mode, just provide a list of PDB IDs or local file names, e.g.:
plip -i 1vsn 1osn 2reg -vxPLIP will create subdirectories for each given structure in the output folder.
If in PDB ID mode (i), the folder structure will be nested and based on the two middle characters of the PDB ID.
The structure 1vsn in batch processing will have its output files in /vs/1vsn .
For the detection of ligands, PLIP relies on the separation of ATOM and HETATM entries in the PDB file.
The latter are searched for suitable ligands when running in normal mode.
Peptide ligands, however, are usually deposited as ATOM entries in a separate chain.
PLIP can not detect these entities automatically.
To switch into protein-peptide interaction mode, start PLIP with the option --peptide, followed by the peptide chain of interest, e.g.:
plip -i 5hi4 --peptides I -vxIntra-protein interactions are important for the stabilization of a structure and can give valuable insights for protein engineering and drug discovery.
PLIP now supports detection of interactions within one chain.
To switch into intra-chain interaction mode, start PLIP with the option --intra, followed by the protein chain of interest, e.g.:
plip -i 5b2m --intra A -yvPlease note that detection within a chain takes much longer than detection of protein-ligand interactions, especially for large structures.
PLIP can characterize interactions between ligands and DNA/RNA.
A special mode allows to switch from protein to DNA/RNA as the receptor molecule in the structure.
To change the receptor mode, start PLIP with the option --dnareceptor.
The PLIP algorithm uses a rule-based detection to report non-covalent interaction between proteins and their partners. The current settings are based on literature values and have been refined based on extensive testing with independent cases from mainly crystallography journals, covering a broad range of structure resolutions. For most users, it is not recommended to change the standard settings. However, there may be cases where changing detection thresholds is advisable (e.g. sets with multiple very low resolution structures) PLIP allows you to change the setting permanently or for one run.
PLIP settings are stored in the file modules/config.py, which is loaded in each run.
One section in the file includes assignments of standard values to all distance and angle thresholds, together with short descriptions.
Changing the values in this file means they will be changed permanently for PLIP.
Furthermore, filter settings for ligands (metal ions in complexes, unsupported ligands, warnings for possible artifacts) can be changed in this file as well.
Thresholds can be changed for each run using command-line parameters. The naming of the variables is identical to those in the config file, but all lowercase. Specify the threshold you want to change and the new value, e.g. to change HYDROPH_DIST_MAX to 5 Angstrom, run PLIP using
plip -i 1vsn --hydroph_dist_max 5All distance thresholds can be increased to up to 10 Angstrom. Thresholds for angles can be set between 0 and 180 degree. If two interdependent thresholds have conflicting values, PLIP will show an error message.
PLIP offers further command line options which enables you to switch advanced settings.
- Set number
nof maximum threads used for parallel processing (--maxthreads <n>) - Do not automatically combine covalently bound ligands (
--breakcomposite) - Do not discard alternate locations (
--altlocation) - Set debug mode (
--debug) - Turn off automatic fixing of errors in PDB files (
--nofix) - Keep modified residues as ligands (
--keepmod)
A web service for analysis of protein-ligand complexes using PLIP is available at http://plip.biotec.tu-dresden.de/ The web site offers advanced functions to search for specific entries from PDB and lists the interaction results in the browser. Additionally, the service used the BioLiP database to annotate biologically relevant ligands. The option to change threshold, ligand filtering, and batch processing is only available in the command line tool and with the Python modules.
PLIP uses a rule-based system for detection of non-covalent interactions between protein residues and ligands. Information on chemical groups able to participate in a specific interaction (e.g. requirements for hydrogen bond donors) and interaction geometry (e.g. distance and angle thresholds) from literature are used to detect characteristics of non-covalent interactions between contacting atoms of protein and ligands. For each binding site, the algorithm searches first for atoms or atom groups in the protein and ligand which could possibly be partner in specific interactions. In the second step, geometric rules are applied to match groups in protein and ligand forming an interaction.
Previous to the detection step for the interactions, PLIP extracts all ligands contained in the structure. Modified amino acids are identified and excluded using MODRES entries of the PDB files. Additionally, it uses the BioLiP list of possible artifacts to remove ligands which are in this list and appear 15 times or more in a structure. Just a few compounds are currently excluded, being listed in the PLIP config file.
Polar hydrogens are added to the structure and alternative conformations/models/positions removed. Missing chains are assigned to ligands and non-standard ligand names (with special characters) altered to LIG.
The binding site distance cutoff is determined by adding up BS_DIST_MAX to the maximum extent to the ligand (maximum distance of a ligand atom to ligand centroid). All protein atoms within this distance cutoff to any binding site atoms are counted as belonging to the binding site.
An atom is classified as hydrophobic if it is a carbon and has only carbon or hydrogen atoms as neighbours.
OpenBabel is used to identify rings (SSSR perception) and their aromaticity. In cases where no aromaticity is reported by OpenBabel, the ring is checked for planarity. To this end, the normals of each atom in the ring to its neighbors is calculated. The angle between each pair of normals has to be less than AROMATIC_PLANARITY. If this holds true, the ring is also considered as aromatic.
OpenBabel is used to identify hydrogen bond donor and acceptor atoms. Halogen atoms are excluded from this group and treated separately (see below).
The detection of charged groups is only exhaustive for the binding site, not the ligands. For proteins, positive charges are attributed to the side chain nitrogens of Arginine, Histidine and Lysine. Negative charged are assigned to the carboxyl groups in Aspartic Acid and Glutamic Acid. In ligands, positive charges are assigned to quaterny ammonium groups, tertiary amines (assuming the nitrogen could pick up a hydrogen and thus get charged), sulfonium and guanidine groups. Negative charges are reported for phosphate, sulfonate, sulfonic acid and carboxylate.
Assuming that halogen atoms are not present in proteins (unless they are artificially modified), halogen bond donors are searched for only in ligands. All fluorine, chlorine, bromide or iodine atoms connected to a carbon atom qualify as donors. Halogen bond acceptors in proteins are all carbon, phosphor or sulphur atoms connected to oxygen, phosphor, nitrogen or sulfur.
Water atoms are assigned to a ligand-binding site complex if their oxygen atoms are within a certain cutoff to the ligand. The cutoff is determined by adding up BS_DIST_MAX to the maximum extent to the ligand (maximum distance of a ligand atom to ligand centroid). This means the farthest distance of a ligand to a water atom is BS_DIST_MAX.
For an overview on geometric cutoffs used for the prediction of interactions, please refer to the config file. Note that the threshold can not be changed for jobs running on PLIP Web. The command line tool (sourcecode available for download), however, allows changing all listed parameters permanently or for single runs.
As hydrophobic interactions result from entropic changes rather than attractive forces between atoms, there are no clear geometries of hydrophobic association. The observed attraction between hydrophobic atoms decays exponentionally with the distance between them.
A generous cutoff was chosen, identifying a prime set of hydrophobic interactions between all pairs of hydrophobic atoms within a distance of HYDROPH_DIST_MAX. Since the number of hydrophobic interactions with such an one-step approach can easily surpass all other interaction types combined, it may strongly influence subsequent evaluation or applications as interaction fingerprinting. To overcome this problem, the number of hydrophobic interactions is reduced in several steps. First, hydrophobic interactions between rings interacting via π-stacking are removed. This is done because stacking already involves hydrophobic interactions.
Second, two clustering steps are applied. If a ligand atom interacts with several binding site atoms in the same residue, only the interaction with the closest distance is kept. Subsequently, the set of hydrophobic interactions is checked from the opposite perspective: if a protein atom interacts with several neighboring ligand atoms, just the interaction with the closest distance is kept. Together, these reduction steps help to report only the most representative hydrophobic interactions.
A hydrogen bond between a hydrogen bond donor and acceptor is reported if several geometric requirements are fulfilled. The distance has to be less than HBOND_DIST_MAX and the angle at the donor group (D-H...A) above HBOND_DON_ANGLE_MIN.
Since salt bridges involve purely electrostatic interactions as well as hydrogen bonds, it is not meaningful to report both interaction types between the same groups. Thus, hydrogen bonds between atoms are removed if they belong to groups that already form a salt bridge to that atom.
As a general rule, a hydrogen bond donor can take part in only one hydrogen bond, while acceptor atoms can be partners in multiple hydrogen bonds (e.g. bifurcated hydrogen bonds). For multiple possible hydrogen bonds from one donor, only the contact with the donor angle closer to 180 ° is kept.
π-Stacking for two aromatic rings is reported whenever their centers are within a distance of PISTACK_DIST_MAX, the angle deviates no more than PISTACK_ANG_DEV from the optimal angle of 90 ° for T-stacking or 180 ° for P-stacking.
Additionally, each ring center is projected onto the opposite ring plane. The distance between the other ring center and the projected point (i.e. the offset) has to be less than PISTACK_OFFSET_MAX. This value corresponds approximately to the radius of benzene + 0.6 Å.
π-Cation interactions are reported for each pairing of a positive charge and an aromatic ring if the distance between the charge center and the aromatic ring center is less than PICATION_DIST_MAX. In the case of a putative π-cation interaction with a tertiary amine of the ligand, an additional angle criterion is applied (see documentation in the source code).
Whenever two centers of opposite charges come within a distance of SALTBRIDGE_DIST_MAX, a salt bridge is reported. In contrast to hydrogen bonds, there are no additonal geometric restrictions.
While residues can be bridged by more than one water molecule, for the prediction in this script the only case considered is one water molecule bridging ligand and protein atoms via hydrogen bonding.
The water molecule has to be positioned between hydrogen bond donor/acceptor pairs of ligand and protein with distances of the water oxygen within WATER_BRIDGE_MINDIST and WATER_BRIDGE_MAXDIST to the corresponding polar atoms of the donor or acceptor groups. If a constellation with a water atom fulfils these requirements, two angles are checked. The angle ω between the acceptor atom, the water oxygen and donor hydrogen has to be within WATER_BRIDGE_OMEGA_MIN and WATER_BRIDGE_OMEGA_MAX. Additionally, the angle θ between the water oxygen, the donor hydrogen and the donor atom has to be larger than WATER_BRIDGE_THETA_MIN.
Similar to standard hydrogen bonds, a water molecule is only allowed to participate as donor in two hydrogen bonds (two hydrogen atoms as donors). In the case of more than two possible hydrogen bonds for a water molecule as donor, only the two contacts with a water angle closest to 110 ° are kept
Halogen bonds are reported for each pairing of halogen bond acceptor and donor group having a distance of less than HALOGEN_DIST_MAX and angles at the donor and acceptor group of HALOGEN_DON_ANGLE and HALOGEN_ACC_ANGLE with a deviation of no more than HALOGEN_ANG_DEV
For metal complexes, PLIP considers metal ions from a set of more than 50 species (see PLIP config for more details). Possible interacting groups in the protein are sidechains of cystein (S), histidine (N), asparagine, glutamic acid, serin, threonin, and tyrosin (all O), as well as all main chain oxygens.
In ligands, following groups are considered for metal complexation: alcohols, phenolates, carboxylates, phosphoryls, thiolates, imidazoles, pyrroles, and the iron-sulfur cluster as a special constellation. For one metal ions, all groups with a maximum distance of METAL_DIST_MAX to the ligand are considered for the complex.
After assigning all target groups to one metal ions, the resulting set of angles of the complex is compared with known sets of angles from common coordination geometries (linear [2], trigonal planar [3], trigonal pyramidal [3], tetrahedral [4], square planar [4], trigonal bipyramidal [5], square pyramidal [5], and octahedral [6]). The best fit with the least difference in observed targets is chosen as an estimated geometry and targets superfluous to the constellation are removed.
| Exit code | Description |
|---|---|
| 1 | Unspecified Error |
| 2 | Empty PDB file as input |
| 3 | Invalid PDB ID (wrong format) |
| 4 | PDB file can't be read by OpenBabel |
| 5 | Valid PDB ID, but no PDB file on wwwPDB |
| Description | RGB | PyMOL color | Representation |
|---|---|---|---|
| Protein | [43, 131, 186] | myblue (custom) | sticks |
| Ligand | [253, 174, 97] | myorange (custom) | sticks |
| Water | [191, 191, 255] | lightblue | nb_spheres |
| Charge Center | [255, 255, 0] | yellow | spheres |
| Aromatic Ring Center | [230, 230, 230] | grey90 | spheres |
| Ions | [250, 255, 128] | hotpink | spheres |
| Description | RGB | PyMOL color | Representation |
|---|---|---|---|
| Hydrophobic Interaction | [128, 128, 128] | grey50 | dashed |
| Hydrogen Bond | [0, 0, 255] | blue | solid line |
| Water Bridges | [191, 191, 255] | lightblue | solid line |
| pi-Stacking (parallel) | [0, 255, 0] | green | dashed line |
| pi-Stacking (perpendicular) | [140, 179, 102] | smudge | dashed line |
| pi-Cation Interaction | [255, 128, 0] | orange | dashed line |
| Halogen Bond | [54, 255, 191] | greencyan | solid line |
| Salt Bridge | [255, 255, 0] | yellow | dashed line |
| Metal Complex | [140, 64, 153] | violetpurple | dashed line |
report
Contains all binding site information
plipversion
Version of PLIP used for generating the output file
bindingsite
Information for one bindingsite. Has a unique ID and attribute has_interactions
identifiers
Ligand/bindingsite identifiers
longname
Long name of ligand, contains all het ids of ligands in one composite cluster
ligtype
Classification of ligand, can be SMALLMOLECULE/POLYMER/DNA/RNA/ION or combinations of the first four with ION
hetid
PDB hetero ID of the ligand
chain
Chain assigned to the ligand in the PDB file
position Position in chain of the ligand in the PDB file
composite
Can be True or False depending on whether the ligand consists of several separate subunits or not
members
Lists the members of a composite ligand cluster
smiles
The SMILES string of the complete (composite) ligand
lig_properties
Additional information on the ligand, i.e. number of functional atoms
num_heavy_atoms
Number of heavy atoms in the ligand
num_hbd
Number of hydrogen bond donors in the ligand
num_unpaired_hbd
Number of unpaired hydrogen bond donors in the ligand (not involved as acceptor/donor in hydrogen bonds, salt bridges, water bridges, metal complexes)
num_hba
Number of hydrogen bond acceptors in the ligand
num_unpaired_hba
Number of unpaired hydrogen bond acceptors in the ligand (not involved as acceptor/donor in hydrogen bonds, salt bridges, water bridges, metal complexes)
num_hal
Number of halogen bond donors in the ligand
num_unpaired_hal
Number of unpaired halogen bond donors in the ligand
num_aromatic_rings
Number of aromatic rings in the ligand
interacting chains
Lists the chains the ligand interacts with
bs_residues
Listing of binding site residues the ligand is near to or interacts with. Contains the type of amino acid, information on contact, a unique id and the minimal distance to the ligand in Angstrom
interactions
Detailed information on all interactions (general attributes documented below)
resnr
Residue number of interacting amino acid
restype
Residue type of interacting amino acid
reschain
Residue chain of interacting amino acid
resnr_ligand
Residue number of interacting ligand residue
restype_ligand
Residue type of interacting ligand residue
reschain_ligand
Residue chain of interacting ligand residue
dist
Distance of interacting atoms or groups in Angstrom
ligcoo
Coordinates of interacting ligand atom or interaction center in ligand
protcoo
Coordinates of interacting protein atom or interaction center in ligand
To run the tests, run the following set of commands.
cd ~/pliptool/plip/test
python -m unittest test*