FEP+ — Analysis Panel

Display detailed information on the protein-ligand interactions, ligand properties and changes, protein changes, and simulation convergence for a complex or for two complexes that are connected by a selected connection. The connection can be for a ligand mutation or a protein mutation. The title bar identifies the mutation. The information is given for the end points of the perturbation, λ = 0 and λ = 1. No information is given for intermediate stages.

To open this panel, click the View button in a row of the Analysis Tab, or right-click on a connection in the Map Tab and choose Analyze, in the FEP+ Panel.

  • Features
  • Additional Resources

Analysis Panel Features

Some of the features in this panel depend on whether the analysis is made for an edge (relative binding FEP) or a complex (absolute binding FEP).

Summary tab

This tab displays a summary of the perturbation, showing the 2D structures of the ligands, marked up according to the selected property.

Ligand 2D structures

Shows the 2D structures of the two ligands, annotated with the selected property. The ΔΔG for the connection is displayed between them, with an arrow indicating the direction of the change.

Overlays options

Select the atom properties to display on the 2D structures. Only present for ligand mutations.

  • Mutation—highlight the mutation. The mutated atoms are marked with red circles and the changed bonds are colored red.
  • Common core—Mark the common core with green bonds.
  • RMSF—Atoms are annotated with colored circles depending on the RMSF value. A legend is shown on the bottom. Use the Type option menu to change the type of RMSF to show.
Show atom mapping option

Show the atom numbering on the structures.

Protein-Ligand Interactions tab

This tab shows information on the interactions between the protein and the ligands. The protein-ligand contacts are classified into four types, whose definitions are given under Contact options in the Simulation Interactions Diagram topic. When an edge is analyzed, there are two figures or two lines on the graph, one for each ligand.

Interaction Diagrams tab

Show a ligand interaction diagram for each complex. Only present for ligand mutations.

Diagram

Residues are represented as colored spheres, labeled with the residue name and residue number, and colored according to their properties. The ligand is displayed as a 2D structure. Interactions between the residues and the ligand are drawn as lines, colored by interaction type, and labeled with the contact strength. Residues that are present but don't have a line drawn for an interaction are involved in nonspecific hydrophobic interactions with the ligand.

Minimum contact strength slider and box

Specify the minimum contact strength for a residue to be displayed in the diagrams. The contact strength is the percentage of time that the residue is in contact with the ligand during the course of the simulation.

Bar Charts tab

Show bar charts indicating the percentage of time that the ligands are interacting with the protein, or an interaction energy breakdown for the residues.

Show options

Specify the interactions to show in the bar charts:

  • Protein-Ligand interactions—show the total percentage of the simulation time that the ligand, or each ligand, is interacting with the protein.
  • Energy decomposition—show the interaction energy of the residues, separated into Van der Waals and Electrostatic energies.

Note: Energy decomposition is not available for non-combined protein FEP workflows, which will be deprecated in the 25-3 release.

Bar charts

This area displays the charts. For a complex, the chart has the residues on the horizontal axis and the interaction percentages or energies on the vertical axis. The two ligands are plotted side by side for each interacting residue. You can use the ligand options on the lower right to show or hide that ligand in the chart. Hovering on a bar displays a tooltip with residue information and a breakdown of the interaction percentages or energies.

When Protein-Ligand interactions is selected, bars show the total percentage of interaction time. The percentages can exceed 100% because the ligand can form multiple interactions with the same residue. So, if two ligand sites interact with the residue, the total percentage can be up to 200%.

When Energy Decomposition is selected, bars show the Van der Waals and Electrostatic components of the interaction energy. The chart only displays residues whose total absolute interaction energy [(abs(Van der Waals) + abs(Electrostatics)] is above 2.0 kcal/mol.

 

Interaction types options

Choose the types of interactions to display by clicking on the colored check box, which reflects the color of the interaction in the bar chart.

For Protein-Ligand interactions, there are five options: H-bonds, Ionic, Water bridges, Hydrophobic, and Halogen bonds.

For Energy Decomposition, there are two options: Van der Waals and Electrostatics.

Ligands options

Choose which ligands to display by clicking on the grey check box.

Ligand Details tab

This tab shows details of ligand properties derived from the simulation. For edge analysis, the plots have two lines, or there are two charts, one for each ligand or protein variant.

Properties tab

This tab shows a plot of the RMSD of the ligand in the complex, and a selection of properties of the ligand in solution and in the complex, with error estimates derived from the simulation. When you move the pointer over the plot area, a vertical line is displayed at the time position, and the time and RMSD values are displayed to the right of the line.

The RMSD of a ligand is measured by first aligning the protein-ligand complex on the protein backbone of the input protein structure, and then measuring the RMSD of the ligand heavy atoms. Note that the RMSD values can fluctuate more than you typically observe in regular MD simulations: large values merely reflect multiple conformations that can exist in the neighboring replicas.

A summary table with data for each ligand is shown at the bottom of the tab.

Rotatable Bonds tab

This tab shows plots for the rotatable bonds in the ligand. The information for ligand in solvent is absent for protein mutation. For each rotatable bond, a representative dihedral angle is monitored throughout the simulation for both complex and solvent legs. The values of the dihedral angle for all the time steps are binned and plotted as a bar chart, along with the torsional potential. The bars for the complex are colored, while those for the ligand in solvent have a white background. The bars also have diagonal stripes going in different directions for the complex and the solvated ligand, so that you can distinguish them when they overlap. A gray vertical line marks the initial value of the rotatable bond angle, which comes from a separate calculation run outside of the simulation .

The strain in kcal/mol is reported below the plot for the complex and solvent legs. The strain is the average of the torsion potential value over all frames in the FEP+ simulation. Large differences in the strain energies between the two legs may indicate a large strain energy or insufficient sampling.

The 2D structures of the ligands are displayed at the top, with the rotatable bonds marked. When you move the pointer into the plots, the other atoms that define the dihedral angle are highlighted on the ligands.

The settings menu allows you to normalize the plots (Normalize Y-Axes) so that the maximum value on the vertical axis is the same for all plots, and set this maximum value (Set Max Y Value).

Protein Details tab

This tab displays details of protein changes during the simulations for the complex or complexes. The RMSD and RMSF are calculated after superimposing the protein backbone for a given time step on the reference frame (the initial structure).

RMSD tab

This tab displays the RMSD of the backbone atoms with respect to the initial position during the course of the simulation. When you move the pointer over the plot area, a vertical line is displayed at the time position, and the time and RMSD values are displayed to the right of the line.

If the simulation has equilibrated, the protein RMSD should be fluctuating around some thermal average, by around 1-3 Å. If the RMSD is still increasing or decreasing, the simulation has not equilibrated, and the simulation may not be long enough for rigorous analysis. Large changes in the protein RMSD may indicate conformational changes.

RMSF tab

This tab displays root-mean-square fluctuations (RMSF) for each residue in the protein chain. The RMSF for the residues is the time-averaged fluctuation of the square deviation of a designated set of residue atoms over the entire simulation time, after superposition on the reference frame. Peaks indicate areas of the protein that fluctuate most. These usually include the termini. Helices or strands usually fluctuate less.

RMSF display

The plots of the RMSF as a function of simulation time, and other markers, are displayed in this area. When you move the pointer over the plot area, a vertical line is displayed at the residue position. The chain, residue number and name are displayed to the left of the line, and the RMSF values and B-factor are displayed to the right of the line.

Show options

Select options for information to show in the plot.

  • Ligand contacts—Mark protein residues that are in contact with the ligand or ligands. The three sub-options for edge analysis allow you to select contacts that are common to both ligands or proteins or unique to one or other of the ligands or proteins.
  • Secondary structure—Highlight the regions on the diagram that correspond to alpha helices (in red) or beta strands (in blue). The regions are defined by helices or strands that persist for more than 70% of the simulation.
  • B-factor (PDB)—If the protein has experimental B factors, these are plotted on the diagram. The RMSF should correlate fairly well with the B factors.

Convergence tab

This tab displays information on the free energy convergence. Free energy differences given in kcal/mol in each leg of the calculation are plotted as a function of time. Three plots for each leg show the accumulated data during different time window schemes: forward; reverse; and sliding window.

Rest Sampling tab

This tab displays charts showing the replicas and how they are exchanged between lambda windows. There are two subtabs, one for the complex/fragment leg and one for the solvent leg.

Each replica is color coded and the plot shows how it occupies different lambda windows during the course of the simulation. The height of a color block for a given replica and lambda window represents the fraction of time the replica spent in the lambda window. Ideally each replica will sample all lambda windows uniformly; however non-uniform sampling is sufficient in most instances.

Generate PDF Report button

Generate a PDF file that reports all of the information presented in the panel, with plots, and includes explanatory text. Opens a file selector, so you can navigate to a location and name the file.

Save Image button

Save an image of the plot in the current tab, in PNG or JPEG format. Opens a file selector, so you can navigate to a location and name the file. When saving an image from the Interaction Diagrams tab, two images are saved, with _1 and _2 appended to the file name provided.

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