Results interpretation

CaverDock results

To fully understand CaverDock results, one needs to be familiar with the basic principles of CaverDock’s method, which is used to analyze a ligand’s movement through a tunnel. The method uses restrained docking, with two types of restraints:

• placement of a ligand atom at the 2D disc in 3D space;

• placement of the whole ligand in upper-bound vicinity of the defined snapshot.

First, CaverDock computes the so-called lower-bound trajectory. With this trajectory, a ligand atom (closest to the ligand centre by default) is iteratively placed and consequent discs. This method samples the tunnel finely; however, a ligand trajectory may contain discontinuities (a ligand can e.g. flip when moved to a different disc). Thus, some bottlenecks may not be detected. Second, CaverDock starts to search for a contiguous trajectory by docking to consequent discs, but always in the vicinity of the previous snapshot.

Snapshots of the lower-bound trajectory are computed for each disc separately; thus, the trajectory search space is relatively small. On the other hand, the position of each snapshot of the contiguous trajectory depends on the previous snapshot. As there are exponentially many trajectories with respect to the number of discs, an exhaustive search of all contiguous trajectories is not feasible. CaverDock uses the heuristic method to prune the space of possible trajectories. The heuristic does not guarantee that the trajectory with the lowest energy is found; thus, we call a contiguous trajectory upper-bound trajectory, as it upper-bound energy of the transport process (the actual energy is the same or lower).

Ideally, the lower-bound and upper-bound energies approximately agree. If the difference is higher than expected, we need to explore the trajectory and examine whether the bottleneck which appeared in the upper-bound trajectory is rather an artefact of the computational method, or if it is created by a real barrier in the tunnel. We can also try to improve the precision of the CaverDock computation using the procedure described in the section Best practices.

Analysing the energy profiles

In the schematic energy profile below we show the important energy values which describe any CaverDock trajectory. The marked energy values are: E_Bound, the binding energy of a ligand located inside the active site; E_Max, the highest binding energy in the trajectory; E_Surface, the binding energy of the ligand located at the protein surface; E_a (k_on), the activation energy of dissociation for the products (E_Max - E_Bound) and E_a (k_off) the activation energy of association for the reactants (E_Max - E_Surface), corresponds to the kinetics of a ligand passing through the tunnel; \(\Delta\) E_BS, the difference of the binding energies in the bound state and at the surface corresponds to the enthalpy of binding, and is related to the equilibrium constant.

You will need to set up the variables for saving specific parts from the parsed energy profile. As a guideline for automatised analysis, we recommend the following ranges for saving the energies: Binding site (bound) - the first half of the profile; Maximum part of the profile (max) - will be used to find and save the profile maximum, use either the whole energy profile or you can, for example, omit the first tenth of the profile if you see peaks in the plot which could be caused by collisions of the molecule with the bottom of the active site, also you can strictly specify the part of the profile representing the barrier. Surface (surface) - you can use the value from the last disc or set your range at the end of the profile.

The energy analysis can be set up either by using specific integer disc numbers (DISC_NUMBER) or float number fractions (FRACTION) of the profile for a more general approach. If the disc ranges are not specified by either numbers or fractions, the analysis will be run with default settings based on the recommendations above.

Command line (Apptainer and prebuilt)Python API

$ cd-analyseeprofile -d IN -s LOWERBOUND -b DISC_NUMBER -o results.csv \
    --bound-start 0 --bound-end 10 \
    --max-start 10 --max-end 100 \
    --surface-start 90 --surface-end 100 \
    profile1.dat profile2.dat