# Tutorials¶

These are tutorials about how to use specific floes.

## Short Trajectory MD with Analysis¶

The Floe Short Trajectory MD with Analysis (STMD) is for pose validation. You should start with a ligand with one or more conformers already well posed in the active site, with protein-ligand interactions in place, serving as hypotheses for ligand binding. Each pose will be evaluated by this floe to be validated (or not) as a good pose. We are not primarily asking the question “what is the pose for this ligand?” but rather “is this a good pose for this ligand?”, or with multiple poses, “which of these poses is best?” We seek an answer by running a short MD trajectory on each pose separately and comparing the results to the starting (input) pose and between the poses (if more than one).

### What this Floe does¶

The structure of the STMD floe is shown in figure Structure of the STMD floe. Given the inputs of the protein and posed ligands, the complex is formed with each ligand/conformer separately, and the complex is solvated and parametrized according to the selected force fields. We refer to this ready-to-run molecular assembly as a “flask” by analogy to experiment: all the components are combined into the flask, upon which we run our experiment. A minimization stage is performed on the flask followed by a warm up (NVT ensemble) and several equilibration stages (NPT ensemble). In the minimization, warm up, and equilibration stages, positional harmonic restraints are applied on the ligand and protein. At the end of the equilibration stages, a short (default 2ns) production run is performed on the unrestrained flask. The production run is then analyzed in terms of interactions between the ligand and the active site and in terms of ligand RMSD, after fitting the trajectory based on active site C_alphas.

Structure of the STMD floe

### Ligand Input¶

Just to be able to run, this floe requires ligands to have reasonable 3D coordinates, all atoms, and correct chemistry (in particular bond orders and formal charges). If the ligands already have good atomic partial charges (we recommend RESP or AM1-BCC_ELF10 charges), we recommend using these for STMD as opposed to re-charging them in the STMD floe.

Given that this floe only runs a very short timescale by default (2 ns), it is preferable that the input pose be well refined.

Although bad clashes (or poor positioning for interactions which you know are important) can be (and often are) cleaned up by even this short trajectory, it starts off the “evaluation” purpose of the floe on the wrong foot by giving a poor comparator.

Poor initial poses might even be considered outside the scope of this floe, given how short is the default timescale. This is why we strongly recommend that docked poses be subsequently minimized in the active site before input to STMD. This will resolve high gradients (usually clashes) with the protein and to allow protein-ligand interactions to optimize in the context of a good force field. It is possible that even with this pre-MD refinement, the docked-pose starting points could be reevaluated and triaged prior to the extra effort and expense of STMD. The ligand input datasets used in this tutorial are:

### Protein Input¶

All the MD floes require correctly prepared protein up to “MD ready” standards. This begins with the normal prerequisites for physics-based modeling:

• Protein chains must be capped,

• All atoms in protein residues (including hydrogens) must be present, and

• Missing protein loops resolved or capped.

Of course, protein side chain formal charges and protonation at this point determine their tautomeric state.

Additionally, cofactors and structured internal waters are also important to include, not only those in the immediate vicinity of the ligand and active site but also distally because they can have an important effect on the protein structure and dynamics over the course of the MD.

We strongly recommend using Spruce for protein preparation. The protein input dataset used in this tutorial is:

Warning

Unfortunately, proteins with covalently bound ligands or covalently bound cofactors are currently not tractable

### How to use this floe¶

After selecting the Short Trajectory MD with Analysis floe in the Orion UI, you will be presented with a job form with parameters to select. In Figure STMD Job Form for ligand 35 (1 pose) you can see how we filled out the key fields of that form for the ligand 35 1-pose case described below.

Key fields of STMD Job Form for ligand 35 (1 pose)

Aside from the essential user-defined parameters relating to jobname, input (protein and ligand datasets as described above), and output (output and failure dataset names), all other parameters except “Flask_title” have reasonable defaults. This example is for an MCL1 protein, so after setting “Flask_title” to “MCL1”, launching the floe at this point is fine. That said, the top-level parameters you may consider changing are:

• Flask_title (no default): Here is where you can put a handy short name for the protein to use in molecule titles (e.g. “Bace” instead of “beta-secretase”).

• N_md_starts (default 1): This allows the user to ask for N independent starts to each ligand/pose, giving rise to N independent MD runs; this gives more sampling while keeping the simulation closer to the starting pose.

• Charge_ligands (default True): If your input ligands already have good atomic partial charges (e.g. RESP or AM1-BCC_ELF10), set this to False to have the floe use the existing ligand charges.

• Ligand_forcefield (default OpenFF1.3.1): This forcefield choice has a strong impact on the results. We recommend the most recent version of the OpenFF force field from the Open Force Field Initiative.

• Md_engine (default OpenMM): Gromacs is the other alternative but we recommend OpenMM because HMR works with it but not yet with Gromacs.

• Hmr: Hydrogen Mass Repartitioning (HMR) gives a two-fold speedup and reduces cost. We recommend leaving it on.

We make the other top-level parameters available for expert users by turning on “Show Cube Parameters” at the bottom of the input form and then drilling down into the parameters of the desired cube in the list below.

### Accessing and Understanding the Results¶

The results from the STMD floe are accessed via two main avenues: through the job output in the Jobs tab in Orion’s Floe page, and through orion’s Analyze page. We will look at the results of two jobs run on the same MCL1 ligand; in the first case the input ligand had only a single pose and in the second case it had six slightly different docked poses.

### MCL1 ligand 35: single input pose¶

First we will look at the results of the single-pose run, with default of 1 for N_md_starts: one start of one ligand with one pose, so one 2 ns MD run overall. In the Jobs tab in Orion’s Floe page, having selected the job name for your STMD job, you should land on the job results page. The left panel contains the usual orion job information from the run, and the right panel has one tab at the top if the run was not successful or two tabs at the top if it was… we will focus on success here! Selecting the second tab called FLOE REPORT should give you a page looking similar to Figure STMD Job results page for a single pose of an MCL1 ligand.

STMD Job results page for a single pose of an MCL1 ligand

The floe report shows a tile for each MD simulation, here there was only one ligand in the input file. The atom colors correspond to calculated B-factors, similar to Xray B-factors, depicting the mobility of those atoms in the active site over the course of the MD trajectory. This gives an immediate read-out on how much various fragments of the ligand were moving around in the active site. As a general principle greater movement suggests that that fragment is not as tightly bound in the active site, but inferences are only qualitative. Certainly fragments hanging out in water of even a tightly bound inhibitor will be expected to be more mobile than the buried parts. Other information on each tile is:

• The ligand name.

• The number of clusters formed by clustering the ligand positions in the MD trajectory.

• The Boltzmann-weighted MMPBSA score for ligand binding over the trajectories for all poses.

• The simple ensemble average BintScore for ligand binding over the trajectories for all poses (lower score is better).

• The stability of the pose relative to the starting pose (varies between 0 (no stability) and 1 (completely stable)).

Clicking on the tile drills down into the detailed analysis of that simulation, resulting in Figure Detailed results for ligand 35 (single pose):

Detailed results for ligand 35 (single pose)

In the graphic we see a 2D representation of the ligand binding interactions for the whole trajectory, with the default display of the Overall tab at the top of the graphic. It is an interactive graphic: selecting the Cluster 0 tab in blue will change the binding interaction representation to that corresponding to the selected cluster. Hovering over one of the interaction in the diagram lights up a strip chart on the right-hand side grey arrow showing the occupancy of that interaction over the course of the trajectory. Within the heavy frame of the graphic, we see that the interactive graph is on interactions; selecting torsions changes the depiction to show a heavy black dot in each rotatable bond. Hovering over one of these shows a radial bar graph of the occupancy of the torsion on the right-hand side. Selecting B-factor yields a depiction of the calculated B-factors for the selected cluster as in the parent tile, but additionally shows the calculated B-factor for each active site amino acid close to the ligand. To the left of the graphic is information about the clustering of the ligand trajectory, including a table giving the ensemble average MMPBSA energy and BintScore (each with standard error) for each cluster. The MMPBSA value represents a Boltzmann-weighted average over all major clusters, But for BintScore it is a simple ensmble average for the ligand as a whole. Note with only one cluster here, the Boltzmann-weighted result represents cluster 0 completely. The remaining value, “Pose Stability”, is derived from the ensemble BintScore and represents how stable the overall protein-ligand binding interactions are compared to the starting pose (varies between 0 (no stability) and 1 (completely stable)).

Scrolling down exposes a strip chart and two tables detailing relevant analyses of the trajectories for all poses of the ligand. The strip chart for ligand 35 (single pose) is shown in Figure Strip Chart results for ligand 35 (single pose):

Strip Chart results for ligand 35 (single pose)

The strip chart shows a time course over the MD trajectory, maintaining always the same color scheme as in the interactive graphic: blue for cluster 0. Additionally, cluster outliers, which are ligand configurations that do not belong to any cluster, are shown in black. The chart simply shows the cluster occupancy of each frame, telling us that the trajectory spent most of the time in the blue Cluster 0, occasionally sampling outliers. It seems like quite a stable pose!

The two tables below the strip chart, shown in Cluster/Pose information for ligand 35 (single pose) describe a relationship between each cluster found in the MD for the ligand and the starting poses.

Cluster/Pose information for ligand 35 (single pose)

With only one pose used for this run the tables are terse, but below when we look at 6 input poses for the same ligand they will be more informative. The upper table “Cluster RMSD from each Starting Pose” describes how closely each cluster stays to the starting pose: the blue Cluster 0 sticks closely to the initial pose (1.38 Å RMSD). The second table “Cluster Percentage by Starting Pose” simply describes the occupancy that we see in the strip chart: the ligand spends 96% of its time in cluster 0. These figures tells us the blue Cluster 0 is stable and stays close to the initial pose.

### MCL1 ligand 35: 6 input poses¶

Now we will look at the results of another run on the same ligand 35, but this time with 6 different input poses: 3 related poses with the methyl “up” in the upper panel of Figure Input poses for the 6-pose run and 3 related poses with the methyl “down” in lower panel of the same Figure. The “up” and “down” poses are only differentiated in the Figure for clarity; in the input file all 6 poses are together as the 6 conformers of the ligand 35 molecule. Poses 0, 3, and 5 have the methyl “down” and poses 1, 2, and 4 have the methyl “up”… this will be important later. The question we might be asking here is whether the “up” methyl or “down” methyl is preferred, and which of the input poses (if any) is preferred. And of course we want to see if the preferred cluster by MD still retains the binding interactions we thought were good enough to carry ligand 35 along up to this point.

Input poses for the 6-pose run: 3 with the methyl “up” (top) and 3 with the methyl “down” (bottom)

Once the run is completed, again we go to the job results page, not shown here because it is so similar to what we saw with the single-pose example in Figure STMD Job results page for a single pose of an MCL1 ligand (above). Selecting the third tab (“FLOE REPORT”), there is still only a single tile for the single ligand; the results for all 6 poses have been aggregated and analyzed together for that ligand. The atom colors corresponding to the calculated B-factors will often be a lot “hotter” (more red) for multiple-pose inputs because trajectories for diverse poses are aggregated together, often giving higher per-atom fluctuations. Click on the tile to drill down into the detailed analysis, resulting in Figure Detailed results for ligand 35 (6 poses):

Detailed results for ligand 35 (single pose)

The results look quite different from the single-pose case although the binding interactions are mostly the same (the 2D representation shows a different orientation). There are now four major clusters. The table to the right of the graphic gives key information on each cluster. The blue cluster 0 is dominant, accounting for 45% of the trajectory and with the best (lowest) ensemble MMPBSA and Bintscore. Cluster 1 (green) is second largest at 36%, and has less good MMPBSA score and BintScore. Cluster 2 (orange) at only 13% abundance scores the worst compared to the others, while Cluster 3 (pink) scores second best by both MMPBSA and BintScore even though it is the smallest cluster at 5%. How do these clusters relate to the different input poses?

Scrolling down to the strip chart, shown below in Figure Strip Chart results for ligand 35 (6 poses), we see the time course over the MD trajectories for all starting poses concatenated and analyzed together. The strip chart and the table below it (table Cluster Percentage by Pose for ligand 35 (6 poses) both point to a clear grouping by pose: poses 0, 3,and 5 show predominantly cluster 0 occupancy (blue), and poses 1, 2, and 4 show predominantly cluster 1 occupancy (green).

Strip Chart results for ligand 35 (6 poses)

Cluster Percentage by Pose for ligand 35 (6 poses)

The former poses correspond to the methyl “down” starting poses and the latter to the methyl “up” starting poses, which we can confirm in the Orion 3D page. While the short trajectories in this run (2 ns for each pose) do not allow interconversion between methyl “up” and “down” poses, it appears that the 3 poses in each category have collapsed to a single dominant cluster. How close is the cluster to any of the starting poses? This answered by the Table Cluster RMSD from Pose for ligand 35 (6 poses)

Cluster RMSD from Pose for ligand 35 (6 poses)

This table confirms that cluster 0 is quite close to the starting poses (0, 3, and 5) that contributed to it, though slightly closer to Pose 0. Cluster 1 is still within 2 Å of 5/6 poses, but closest to Pose 1 out of all.

We can visually confirm this by selecting the output dataset (in the “Data” tab of Orion) and then going to the “3D” tab. Under the list of structures for ligand 35, the starting poses are conformers under the molecule named simply “35”. Unfortunately the conformer number in this structure are off by 1, starting from “1”, compared to the analysis, which starts from 0! This bug will be fixed in a future release. The average and median for each cluster appear as a separate protein-ligand complex, labeled accordingly (for example “35_clus0_Avg” for the average of cluster 0). Selecting starting poses corresponding to “down” poses 0, 3, and 5 (i.e. conformers 1, 4, and 6) and displaying them with the average for cluster 0 (“35_clus0_Avg”) gives the upper panel in Figure Starting Poses and Cluster Averages for ligand 35. Selecting starting poses corresponding to “up” poses 1, 2, and 4 (i.e. conformers 2, 3, and 5) and displaying them with the average for cluster 1 (“35_clus1_Avg”) gives the lower panel. Interestingly, with the averages color by calculated B-factor it is obvious that the “down” cluster is markedly more stable in the active site than the “up” cluster, as well as being more energetically favorable by MMPBSA and BintScore.

Starting Poses and Cluster Averages for ligand 35

These visually confirm what we had seen emerging from the analysis: the 6 poses collapse into a predominant methyl “up” and methyl “down” pose. Cluster 0 lies close to one of the starting poses, but Cluster 1 lies in between two of the starting poses. Cluster 0 has a more stable pose than Cluster 1, and the ensemble MMPBSA energies and BintScores also favor Cluster 0.

### Analyzing a Set of Ligands¶

Finally we will look at how to visualize the results for a 11-ligand subset that spans the range of activities for the MCL1 dataset. Each of 11 ligands has 5 reasonable input poses from docking. The whole subset will be run in the same job in the “Short Trajectory MD with Analysis” floe. which will consist of 11 ligands * 6 poses each = 66 MD run of 2ns each. Selecting the output dataset in the “Data” tab and moving to the “Analyze” tab, the results for the entire dataset can be viewed at once as in Figure Analyze page for MCL1 dataset:

Analyze page for MCL1 dataset

There are a lot of results showing in this page, encompassing both numerical and 3D information. The 3D info is brought in by selecting Analyze with 3D under the Layout pull-down menu at the top right. The axes of the scatterplot were selected to display the experimental deltaG (included as SD tag ‘r_user_dG.exp’ on the input ligands) on the x axis and the Boltzmann-weighted ensemble MMPBSA value on the y axis. In the 3D visualizer, select ligand 49 and unroll the list of associated structures. The point in the scatter plot corresponding to ligand 49 and the corresponding line in the spreadsheet is highlighted. In the 3D window, the 5 initial input poses for ligand 49 are under Molecule “49” and display in in gold if selected. Turn on the protein-ligand average structures for Clusters 0 and 1, which will be colored by B-factor as before. This way we can compare the poses to the representative average for each cluster, helping us to evaluate and prioritize that ligand. To call up the detailed MD analysis once again, go to the spreadsheet row for ligand 49, and under the column titled Floe_report_URL click on the little square to open up another tab in your browser with the same detailed analysis floe report as we saw above.

There is a lot of information to look at in the results from the Short Trajectory MD with Analysis floe, but this should get you started. We emphasize that a lot of the analyses can only be interpreted qualitatively at this stage, but nevertheless we feel that the sampling of both protein and ligand configurations at physiological temperatures in the context of explicit water solvation can help validate the initial input pose(s).

## Nonequilibrium Switching¶

The Nonequilibrium Switching (NES) method is a relatively novel method in the Binding Free context to calculate Relative Binding Affinities (RBFE) of a given target and its ligands. The theory was developed during the 1990s 1 2. However, due to its high computational demand, the approach has not been fully explored, and few pioneering works have been published so far 3

In general, the relative binding affinity $$\Delta\Delta G$$ is defined as the free energy difference between the binding affinities of a ligand A $$\Delta G_{A}$$ and B $$\Delta G_{B}$$ related to their target: $$\Delta\Delta G=\Delta G_{B}-\Delta G_{A}$$. Nevertheless, the direct computation of these affinities could be quite challenging because a direct binding process has to be simulated in-silico and often, other thermodynamics paths are used. During the last 30 years, Alchemical methods have been proved to be very effective to calculate the RBFE between two ligands (Egde) . In this approach, a starting ligand A is “mutated” into a final one B in different environments. For example, $$\Delta\Delta G$$ can also be computed following the alternative paths shown in the figure below where $$\Delta G_{Bound}$$ and $$\Delta G_{Unbound}$$ are estimated by using alchemical methods: $$\Delta\Delta G=\Delta G_{Bound}-\Delta G_{Unbound}$$.

RBFE and alternative thermodynamics paths

In the Unbound path the starting ligand is mutated into the final one just in solution while, in the Bound path, the mutation happens in the complex binding site. In the NES approach, these mutations are done in a nonequilibrium regime many and many times starting from equilibrium snapshots and building the forward and reverse work probability distribution functions that can be used to estimate $$\Delta G_{Bound}$$ and $$\Delta G_{Unbound}$$. For this reason, the NES methodology requires prior to run to have the equilibrium ensembles for the Bound and the Unbound systems.

In addition, the implemented NES floe-protocol tries to estimate the binding affinities from the computed RBFEs by using the maximum likelihood estimator method 4 and, making possible to compare the predicted affinities values with experimental results derived, for example, from activities measurements, IC50 etc.

Warning

RBFE calculations via Alchemical methods must be carefully used. In particular, mutations should be carried out between similar ligands i.e. where a common ligand scaffold can be identified and small different functional groups are mutated.

### The Equilibrium and Nonequilibrium Switching floe¶

The Equilibrium and Nonequilibrium switching floe can be divided into four main sections shown here.

The four main sections of the Equilibrium and Nonequilibrium Switching floe

The first and second floe sections are designed to assemble and run the Bound and Unbound simulations. The protocol for the Bound simulations is similar to the tutorial on the Short Trajectory MD with Analysis and it is not repeated here. In the Unbound simulations, each ligand is charged, solvated in a box of solvent and parametrized accordingly to the selected force field and then is equilibrated in three different steps performing: restrained minimization, NVT and NPT md runs. At the end of the equilibration stages, both the Bound and Unbound prepared flasks are set into an equilibrium production stage where they run for a total of a default 6ns.

The third section of the floe carries out the NES calculations and is shown here

The Nonequilibrium Switching floe section

In this section, important cubes are the Gathering cube which selects the equilibrium runs involved in a RBFE calculation and handing them to the Chimera cube. In this cube, a chimeric molecule between the ligands participating in an edge is created (the ligand edge are topologically merged together with their force field parameters) and injected into selected equilibrium trajectory frames collected during the equilibrium runs. In the NES method, for each chimeric molecule (or edge) the following switching simulations are performed per each selected equilibrium frame:

• A Bound forward (ligA is mutate to ligB in the complex binding site)

• A Bound reverse (ligB is mutate to ligA in the complex binding site)

• An Unbound forward (ligA is mutate to ligB just in solution)

• An Unbound reverse (ligB is mutate to ligA just in solution)

By default, 80 equilibrium frames are selected for the Bound and Unbound runs therefore, for each edge a total of:

80 (Bound forward) + 80 (Bound reverse) + 80 (Unbound forward) + 80 (Unbound reverse) = 320

ne-switching mutations are completed. Each run is effectively made with a short NPT equilibration of 5ps to adapt the equilibrium frame system to the new chimeric molecule followed by a default 50ps NPT ensemble simulation where the chimeric molecule is switched between the starting and final ligand thermodynamic states. All the runs are done by using Gromacs as md engine at this stage.

The final NES-floe section analyzes the NES data to produce binding affinities and relative binding affinities results.

The NES Analysis floe section

For each edge, the forward and reverse works for the Bound and Unbound switching are evaluated by using the Bennet Acceptance ratio for Nonequilibrium and these values are used to compute the $$\Delta G_{Bound}$$ and $$\Delta G_{Unbound}$$ free energies that are related to $$\Delta\Delta G$$. The RBFE values for an edge are used to attempt estimates of the affinity values by using the maximum likelihood estimator. However, this approach succeeds if the provided ligand edge map is enough connected, otherwise no estimates will be done.

### Protein, Ligand and Edge mapping file inputs¶

As for the Short Trajectory MD floe, the NES floe requires ligands to have reasonable 3D coordinates, all atoms, and correct chemistry. In particular, bond orders and formal charges should be correctly assigned. The floe can be directly input from docking programs like Posit but, bad clashes should have been relaxed prior to input the floe to resolve high gradients with the protein or other components like cofactors. In general, bad poses will evaluated and eventually rejected at floe running-time.

The NES floe requires correctly prepared proteins up to “MD ready” standards which requires chain capping, all atoms in protein residues (including hydrogens) and missing protein loops resolved or capped. Protein side chain formal charges and protonation at this point determine their tautomeric state. Additionally, cofactors and structured internal waters are also important to be included. We strongly recommend using Spruce for protein preparation.

At this stage of the NES development, the floe requires a ligand mapping for the relative binding affinity calculations to be carried out. This is done by using a text file as floe input. This text file has a strict grammar where the first entry of a line is the starting ligand molecule title name, the second entry is the concatenation symbol “>>” and, determines the RBFE calculation or edge direction and, the third entry is the final ligand molecule title name. For example, for the RBFE calculation involving the edge where the ligA has molecule title name “ligA” mutated to the ligaB molecule with title name “ligB” the syntax for this entry into the edge mapping file is:

ligA >> ligB

We are aware that this is not optimal for users and, we are going to easy this step by implementing an automated mapper in further releases.

For this tutorial the Tyk2 receptor and few ligands have been selected. The files can be download below with the ligand mapping text file as well.

### How to use the floe¶

After selecting the Equilibrium and Nonequilibrium Switching floe in the Orion UI, you will be presented with a job form with inputs, outputs and parameters to select. In next the Figures you can see how we filled out the key fields for the Tyk2 receptor case.

The NES floe inputs

The Equilibrium and Nonequilibrium Switching floe requires two mandatory inputs and two optional inputs. The mandatory inputs are the ligand datasets and the ligand edge mapping text file. The two optional inputs are the protein input file and the experimental binding affinity text file. However, If the optional protein is not provided, the floe will check if the protein is present on the ligand datasets in form of Design Unit produced by Spruce. If this data cannot be found an error will be raised and the floe will fail. The OpenEye Posit floe is able to produce datasets in this form otherwise the user must provides a protein as input as well. In the case that the protein is provided as input and, also the protein is present on the ligand input datasets the protein input will supersede. The other optional input is the experimental affinity file which will be used to generate comparison plots and tables between experimental and predicted results for $$\Delta\Delta Gs$$ and $$\Delta Gs$$ in the floe reports.

Warning

The protein input is optional but, if it is not provided the protein must be present on the ligand input datasets as Spruce Design Unit.

In order to submit the floe to Orion output dataset names have to be input to the floe.

The NES floe outputs

The Equilibrium Bound and Unbound dataset outputs are the datasets produced at the end of the Equilibrium runs. The Bound dataset can be further used as input in the Analysis floe for Short Trajectory MD to triage stable from unstable ligand poses. If the provided edge mapping file is well connected, the NES floe can predict Affinities, and these results are saved in the Affinity dataset output. The NES and Failure datasets are also produced as outputs. The first contains all the information produced along the

NES runs at edge and ligand level while, the latter gathers all the failures produce along the whole floe for debugging purposes. Finally, the user must provide a dataset name for the Recovery dataset. Indeed, occasionally, Orion and the AWS cloud infrastructures could have severe problems and the produced recovery dataset can be used to try to recover and generate partial results from the NES runs by using the designed recovery Nonequilibrium Switching Recovery floe.

The final NES floe selection is related to the promoted parameters

The NES floe parameters

Their meanings are explained below:

• Total Number of NES Trajectory Frames (Default 80) This parameters controls how many snapshots are taken

from the Bound and Unbound Equilibrium trajectories to run the NES switching. For example, suppose that in the Bound equilibrium run for 6ns production, we collected a total of 1000 frames. From these frames, 80 equally distanced frames are selected (~each 12 frames). The chimeric molecule is injected into these frames to run the forward and reverse Bound and Unbound runs.

• NES Switching Time (Default 50ps) This parameter controls the time length of the NES switching. For difficult and large mutations, this parameter could be used to try to have better bound/unbound work convergence.

• Protein Title Name (Default blanc) The protein name used to identify your flask. This name will be used for the produced output file names and other information.

• Charge The Ligand (Default True) If True the ligand will be charged by using the ELF10 methods otherwise the ligand partial charged will be used (if any).

• Ligand Force Field (Default OpenFF 1.3) The ligand force field to be used.

• Protein Force Field (Default Amber14SB) The protein force field to be used.

• Hydrogen Mass Repartitioning (Default True) If true the md time step used along the equilibrium runs will be set to 4fs otherwise to 2 fs.

• Equilibrium Running Time (Default 6ns) The total equilibrium time for the Bound and Unbound simulations.

• Ligand Affinity Experimental File (Default None) The experimental text file with the binding affinity in kcal/mol or kJ/mol. The syntax of this text file is strict. Each line entry must be in the syntax form:

• ligA $$\Delta G$$ $$\Delta G_{error}$$ units

where ligA is the molecule title name, $$\Delta G$$ the binding affinity value, $$\Delta G_{error}$$ the binding affinity valuer error and units in the syntax form of kcal/mol or kJ/mol. The $$\Delta G_{error}$$ is optional and if not provided will not be used. An example of this file for the Tyk2 receptor can be downloaded here:

### Accessing and Understanding the Results¶

The results from the floe are accessed via two main floe reports at the end of the running NES job and selectable in the Jobs tab in the Orion Floe page. For this tutorial we will focus on the results produced by running the Tyk2 receptor and ten selected relative binding affinity calculations.

The NES floe finished job

From the NES run job page, the NES Report is accessible by clicking on the tab. Here, many pieces of information are shown, but the most relevant are the edges related to the submitted RBFE calculations in form of tiles. Each tile shows the edge and the predicted RBFE by using the Bennet Acceptance Ratio method for Nonequilibrium. In addition, other relevant pieces of information are shown, like the total floe running time and its cost. Clicking on each tile will show important information related to the RBFE calculation. For example, in the below figure, the detailed calculation information are shown for the edge involving the ligand ejm_46 to ejm_54

A good RBFE calculation

We have two main plots and a table. The upper and lower plots are respectively related to the Bound and Unbound NES simulations. We are going to focus on the Bound NES simulations but, the same considerations are true for the Unbound one. The blue and red graphs are plotting the calculated Forward and Reverse NES switching works for each selected starting equilibrium frames. The important message to take is that if the the graphs overlap well then this is a good indicator that the computed free energy change is trustable and accurate. The probability distribution plots are made by binning the work values for the forward and reverse works and again if the two probability distributions overlap it is a good sign that the calculation was successfully. Analog considerations are valid for the Unbound graph. By using the work values recorded along the switching it is possible to estimate the free energy changes $$\Delta G_{Bound}$$ and $$\Delta G_{Unbound}$$ shown in figure_RBFE and indirectly estimates $$\Delta\Delta G = \Delta G_{Bound} - \Delta G_{UnBound}$$. These computed values are reported in a table as well.

The figure below reports the RBFE calculation involving the ligand ejm_43 to ejm_54

In this case, both the Bound and Unbound graphs and work probability distributions do not overlap well and the results should not be trustable.

From the NES job page, another report, the Affinity Report, is selectable from the tabs. The affinity report shows two main sections. In the first section, experimental and predicted affinities are compared. Here, a graph between $$\Delta G_{Experimental}$$ vs $$\Delta G_{Predicted}$$ is shown. This graph is available if the experimental affinity file has been provided and the ligand edge map is connected enough to be able to make affinity predictions.

Experimental vs predicted affinities

In addition, the graph data are tabulated, and different statistical metrics are shown, such as correlation metrics and linear models. The second section of the Affinity Report shows a comparison between the experimental and predicted relative binding affinities $$\Delta\Delta G_{Experimental}$$ vs $$\Delta\Delta G_{Predicted}$$ in a graph and tables with the statistical metrics as well.

Experimental vs predicted relative binding affinities

References

1

Jarzynski, C. (1997), “Nonequilibrium equality for free energy differences”, Phys. Rev. Lett., 78 (14): 2690

2

Jarzynski, C. (1997), “Equilibrium free-energy differences from nonequilibrium measurements: A master-equation approach”, Phys. Rev. E, 56 (5): 5018

3

Vytautas Gapsys, et al., “Large scale relative protein ligand binding affinities using non-equilibrium alchemy”, Chem. Sci. (2020) 11, 1140-1152

4

Huafeng Xu “Optimal Measurement Network of Pairwise Differences”, J Chem Inf Model. 2019 Nov 25;59(11):4720-4728