PLIP - Help

Detailed Description

Introduction

The Protein-Ligand Interaction Profiler (PLIP) is a web service for analysis of non-covalent interactions in protein-ligand complexes from PDB files. PLIP enables you to choose from existing PDB entries by providing a valid 4-letter PDB code or submit a custom PDB file without further preparation of the structure. After analyzing the complex, the result page lists all detected non-covalent interactions (hydrogen bonds, water bridges, salt bridges, halogen bonds, hydrophobic interactions, π-stacking, π-cation interactions, metal complexes) in atom-level detail. Furthermore, the results are available for download in flat text and machine-readable XML format and for visualization purposes as a PyMOL session file (pse). Within the browser, 3D interaction diagrams can be viewed using embedded JSmol applets.

Input

PLIP Input Form

A There are three options to provide input files for PLIP. If you know a structure from the PDB you want to analyze, just type in the PDB ID of the corresponding Protein Data Bank (PDB) entry. By default, PLIP checks if the given entry is obsolete and replaces the query with the up-to-date structure.

PLIP also allows you to search for a PDB entry by protein structure titles, ligand names (including synonyms), or EC numbers using an integrated wizard (see below for details). Instead of processing a structure from PDB, you can upload your own protein-ligand complex in PDB format, generated e.g. from previous docking or molecular dynamics analyses.

B You are free to provide a job name and mail address in this field. In that case, you will receive a notification as soon as your job has been finished, including a link to the result page

C Run the analysis for the given protein-ligand complex.

PDB Search Wizard

PDB Search Wizard

D To search for protein-ligand complexes in the online PDB archive, type in any combination of protein/ligand name or EC number. The suggestion feature will help you to select search terms.

E Click the Search button to look for entries in the PDB matching your query. PLIP shows the total number of matches F and a list of all hits ranked by importance in relation to the given search terms. In the hit list, the title of the structure as well as HET IDs of ligands in the structure are listed. Matches of search terms are highlighted. Click on a PDB ID to copy it into the search field.

Input File Requirements

PLIP should work for each structure in valid PDB format. As the tool makes use of OpenBabel for processing the files, one option to check the validity is to do a simple conversion with OpenBabel using babel -i pdb <your_file.pdb> -o pdb any_name.pdb If the file can be converted without major errors, it should be ready for use with PLIP. In case of non-standard ligand names or missing chains PLIP performs small fixes to the input file.

Output

Result Summary

PLIP Results Summary

G At the top level of the result page, PLIP groups ligands by type (Small Molecules, Polymer, DNA/RNA, Ions, and combinations thereof) and for each category lists the number and names of all detected ligands interacting with the protein in the given structure. The binding site identifiers consists of the PDB ligand identifier, the chain and the residue assigned to the ligand in the input PDB file. A ligand synonym is given in brackets for most of the compounds in the PDB. As a separate category, all ligands with no interactions are listed (greyed out). For all regular entries, you can click on the binding site identifier to expand to a detailed summary of the protein-ligand interactions (see below). An orange star next to the identifier indicates that the ligand is binding specifically and is thus biologically relevant as defined by the BioLiP database. In the case of composite ligands (e.g. polysaccharides with separate HET IDs for the subunits, or DNA/RNA), the term Composite Ligand is indicated in brackets. The binding site details for those entries lists all members which have been grouped together.

H For further processing or inspection, you can download result files containing interaction data for all ligands in the structure. The text file contains the tables as seen on the result page in a human-readable format. For further computational analysis, you might choose the XML file, which is easily parsable.

I Here, you can choose to return to the input form to run another analysis.

Binding Site Details

PLIP Results Details

J At the top of each binding site entry the identifier and name of the ligand is given (see above). Complexes with composite ligands contain detailed information about the ligand members in red directly below the name.

K For each protein-ligand complex in the structure, PLIP shows a rendered interaction diagram. The ligand is shown in orange, the protein residues in blue color. Non-covalent interactions are indicated by dashed or solid lines as indicated in the legend on the right side. To show an interactive 3D visualization diagram of the interactions in your browser, click on the preview image to open a JSMol applet.

L You can download the rendered image in high resolution or the PyMOL session file which was being used to render the image. In the session file, all structural elements and single interactions are conveniently grouped, allowing you to switch on and off specific interaction types. You can inspect the complex or process it further to generate publication-ready images in a few steps. We recommend using PyMOL 1.7.x. In earlier versions, you might see warnings upon loading the file and encounter missing elements in the visualization or different color schemes. In the case of a red background, you should be able to restore the original color by using set bg_rgb, [1.0, 1.0, 1.0]

M For each interaction type, a table lists the contacts in atomic-level detail. Each entry contains detailed information on protein residues, participating ligand atoms and geometry of the interaction (e.g. distance of interacting atoms). Depending on the type, there is additional information available (e.g. offset of aromatic rings for π-stacking). A detailed explanation for each attribute can be shown by a mouseover on the entry. All atom and residue numbering are in accordance with the numbering in the corresponding PDB file given as input.

PLIP Algorithm

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.

Detection and filtering of ligands

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, we use 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 here.

Preparation of structures

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.

Detection of possible interacting groups

Binding site atoms

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.

Hydrophobic Atoms

An atom is classified as hydrophobic if it is a carbon and has only carbon or hydrogen atoms as neighbours.

Aromatic Rings

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.

Hydrogen Bond Donors and Acceptors

OpenBabel is used to identify hydrogen bond donor and acceptor atoms. Halogen atoms are excluded from this group and treated separately (see below).

Charged Groups

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.

Halogen bonds donors and acceptors

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

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.

Detection of interactions

For an overview on geometric cutoffs used for the prediction of interactions, see also the table below. Please 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.

Hydrophobic Interactions

Hydrophobic Interactions

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.

Hydrogen Bonds

Hydrogen Bonds

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

pi-Stacking

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

pi-Cation Interactions

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).

Salt Bridges

Salt Bridges

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.

Water Bridges

Water bridges

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

Halogen Bonds

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

Metal Complexes

Metal Complexes

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.

Currently unsupported interaction types

  • Covalent bonds
  • Weak hydrogen bonds involving carbon atoms
  • Halogen-Water-Hydrogen Bridges
  • Water bridges of higher degree (bridging over more than one water molecule)
Thresholds used for detection steps in PLIP.
Variable Value Description
BS_DIST_MAX 7.5 Å Cutoff for determination of binding site atoms
AROMATIC_PLANARITY 5.0 ° Cutoff for planarity criterion in aromatic ring detection
HYDROPH_DIST_MAX 4.0 Å Max. distance of carbon atoms for a hydrophobic interaction
HBOND_DIST_MAX 4.1 Å Max. distance between acceptor and donor in hydrogen bonds
HBOND_DON_ANGLE_MIN 100 ° Min. angle at the hydrogen bond donor (D-H...A)
PISTACK_DIST_MAX 7.5 Å Max. distance between ring centers for stacking
PISTACK_ANG_DEV 30 ° Max. deviation from optimum angle for stacking
PISTACK_OFFSET_MAX 2.0 Å Max. offset between aromatic ring centers for stacking
PICATION_DIST_MAX 6.0 Å Max. distance between charge and aromatic ring centers
SALTBRIDGE_DIST_MAX 5.5 Å Distance between two centers of charges in saltbridges
HALOGEN_DIST_MAX 4.0 Å Max. distance between oxygen and halogen
HALOGEN_ACC_ANGLE 120 ° Optimal halogen bond acceptor angle
HALOGEN_DON_ANGLE 165 ° Optimal halogen bond donor angle
HALOGEN_ANGLE_DEV 30 ° Max. deviation from optimal halogen bond angle
WATER_BRIDGE_MINDIST 2.5 Å Min. distance between water oxygen and polar atom
WATER_BRIDGE_MAXDIST 4.0 Å Max. distance between water oxygen and polar atom
WATER_BRIDGE_OMEGA_MIN 75 ° Min. angle between acceptor, water oxygen and donor hydrogen
WATER_BRIDGE_OMEGA_MAX 140 ° Max. angle between acceptor, water oxygen and donor hydrogen
WATER_BRIDGE_THETA_MIN 100 ° Min. angle between water oxygen, donor atom and hydrogen
METAL_DIST_MAX 3.0 Å Max. distance between metal ion and interacting atom