RBFE Tutorial#

Relative Binding Free Energy (RBFE) Workflow with `batter`#

This tutorial walks through a membrane RBFE run powered by batter. The workflow applies a hybrid topology that behaves like dual-topology with a shared core. It uses the simultaneous decoupling/recoupling (SDR) protocol with both ligands present, and relies on softcore electrostatics/van der Waals potentials so the entire calculation completes in a single leg. We reference examples/rbfe_example.yaml so you can reproduce the run locally before adapting it to your own system.

Quick walkthrough#

batter orchestrates an end-to-end AMBER RBFE workflow that starts from protein + embedded protein-membrane system (if applicable) + ligand(s) (3D coordinates) overlaid to the protein binding site. The main steps are:

system staging and loading – An execution folder will be created under <run.output_folder>/executions/ to hold all intermediate files, logs, and results. If a run ID is not provided, a timestamp-based unique ID is generated. If the same run ID already exists, the execution is resumed from the last successful step.
Ligand parameterisation – supports both GAFF/GAFF2 and OpenFF force fields with options to choose charges (AM1-BCC by default)
Equilibration system preparation – builds solvated/membrane-embedded systems with the ligand in the binding site.
Equilibration – Steps to run before FE production run. During this phase, the ligand and protein are not restrained (unless explicitly configured). If the ligand unbinds from the binding site during equilibration, the run is marked as unbound and skipped during FE production.
Equilibrium analysis - Find a representative frame from the equilibrated trajectory to start the FE windows from. RMSD analysis is also performed and saved in the equil folder. Adjust the bound/unbound cutoff via fe_sim.unbound_threshold if your system requires a different distance threshold.
Network planning – Build the RBFE transformation map (pair list) based on the selected scheme.
FE window generation and submission – λ windows are created based on the configuration.
FE equilibration - very short equilibration runs to allow water relaxation. If flag --only-equil is provided, the workflow stops after this step.
FE production runs – Each window runs as an independent local task or scheduler job, depending on how you launch the workflow. The main process monitors job status and streams updates to the terminal. Set run.max_active_jobs in your YAML (default 1000, 0 disables throttling) to cap how many SLURM jobs BATTER keeps active at once and avoid overloading the scheduler.
Analysis – Once all windows complete, MBAR analysis is performed and the final results are written to the portable results/ repository.

Network planning schemes#

RBFE mappings can be created in a few ways:

Default – maps the first ligand to all others (star topology).
Konnektor – uses the konnektor library to build a network; configure with rbfe.mapping: konnektor and optionally rbfe.konnektor_layout. Choose atom mapping backend via rbfe.atom_mapper (kartograf or lomap). The exact Kartograf/LoMap mapper parameters and YAML option blocks are documented in Atom mapper backends. The available layouts are listed in the Konnektor documentation. In BATTER, rbfe.konnektor_layout can be written either as the full class name such as MinimalSpanningTreeNetworkGenerator or as the lowercase shorthand minimalspanningtree. See detailed tutorial in Konnektor tutorial.
Mapping file – provide explicit pairs via rbfe.mapping_file (JSON/YAML list or text file with one pair per line).

Set rbfe.both_directions: true if you want to run both directions for every edge.

Installation#

(Optional) set a persistent pip cache (helpful on shared clusters):
```
export PIP_CACHE_DIR=$SCRATCH/.cache
```

Clone the repository with ssh (or HTTPS if SSH is unavailable) and initialize submodules:

git clone git@github.com:yuxuanzhuang/batter.git
# If SSH is unavailable, use HTTPS instead:
# git clone https://github.com/yuxuanzhuang/batter.git
# For SSH setup tips:
# https://docs.github.com/en/authentication/connecting-to-github-with-ssh/adding-a-new-ssh-key-to-your-github-account

cd batter
git submodule update --init --recursive

Create and activate a Conda environment (with environment.yml):

conda create -n batter python=3.12 -y
conda env update -n batter -f environment.yml
conda activate batter

Install batter itself after the environment update (which already installs the bundled extern/* dependencies):
```
pip install -e .
```
Verify the installation:
```
batter --help
```

Preparing the System#

Use examples/rbfe_example.yaml as the starting configuration. Each field is documented in Configuration Overview, but review the inputs below before running anything:

Required Files#

Protein structure – protein_input.pdb It can be prepared in Maestro or equivalent software. Protonation states are inferred from residue names using AMBER conventions (for example, ASH denotes protonated ASP). When explicit hydrogens are present, BATTER also uses them to distinguish protonation states. Water or non-protein small-molecule coordinates may remain in the file; they are stripped during staging. BATTER currently does not support cofactors or other non-protein residues in protein_input.pdb.
Ligand structures – one ligand per .sdf file with 3D coordinates. Docked poses, aligned experimental structures, or co-folding models all work as long as the coordinates align with the provided protein_input.pdb. Ensure hydrogens/protonation states are correct (Open Babel, the scripts/get_protonation.ipynb notebook, or a similar tool can help). If you use rbfe.atom_mapper: kartograf (the BATTER default), the ligands should preferably be pre-aligned in a consistent binding pose, since well-aligned molecules are one of Kartograf’s core assumptions for finding a good mapping. See the Kartograf mapping tutorial for the upstream guidance.
System topology and coordinates (optional) – system_input.pdb / system_input.inpcrd Needed for membrane protein system.

The membrane-embedded system can be generated via Dabble (preferred with protein_input.pdb). system_input.pdb must encode the correct unit-cell vectors (box information) if system_input.inpcrd is not provided (Dabble does this by default). If system_input.inpcrd is provided its coordinates and box information take precedence.

protein_input.pdb does not need to be aligned to system_input.pdb; it can be helpful in cases e.g., the protein structure used for docking (so all the docked poses are superposed to this protein) is oriented differently from the membrane system. During system staging, the protein will be aligned to the membrane system, and the alignment will be done automatically based on the create.protein_align config setting.

Systems from other builders (CHARMM-GUI, Maestro, etc.) may work but are not extensively tested.

Command to generate POPC-embedded systems with Dabble:
```
dabble -i protein_input.mae -o system_input.prmtop --hmr -w 20 -O -ff charmm
```
In batter preparation process, the membrane molecules will be extracted (controlled by create.lipid_mol); water and ion molecules around create.solv_shell will also be extracted.

Generating Simulation Inputs#

Copy and edit the template. Start from examples/rbfe_example.yaml and save a copy beside your project data. Update:
- run.output_folder – dedicated directory for outputs/logs.
- create.system_name – label used in reports.
- create.ligand_input – JSON file mapping unique ligand IDs to .sdf files (see examples/reference/ligand_dict.json).
- create.* paths – point at your receptor, system, membrane, and restraint files.
- create.anchor_atoms – The three atoms that define the binding site and anchor geometry used during staging and validation. Choose stable backbone atoms (CA/C/N) with the guidelines below.
  
  Anchors (P1, P2, P3) should avoid loop regions, keep P1–P2 and P2–P3 ≥ 8 Å, and target ∠(P1–P2–P3) near 90°.
  
  P1 should preferably form a consistent electrostatics interaction with available bound ligands (e.g., a salt bridge).
  
  For GPCR orthosteric sites, a common choice is P1=3x32, P2=2x53, P3=7x42.
Additional field that may need adjustment based on your cluster environment:
- run.email_on_completion – email address to notify when the BATTER manager finishes or aborts with an uncaught failure.
- run.email_sender – sender address for those notifications. Defaults to nobody@stanford.edu.
- run.slurm.partition – SLURM partition/queue to submit jobs to.
- run.max_active_jobs – cap on how many SLURM jobs to keep active at once (default 1000, 0 disables throttling).
- rbfe.mapping / rbfe.mapping_file – choose your network planning scheme.
- rbfe.atom_mapper – choose RBFE atom mapper backend: kartograf (default) or lomap.
  
  The available schemes are described in Network planning schemes. Mapper options can be overridden under rbfe.kartograf and rbfe.lomap; see Atom mapper backends for the accepted keys and defaults. For mapper-specific behavior and examples, see the Kartograf documentation and the LoMap documentation. As a practical default, start with kartograf unless you have a reason to prefer lomap for a particular ligand series. lomap remains available and can still be a better fit for some chemotypes or mapping preferences.
Use Configuration Overview for the full YAML field reference and RBFE Guide for the RBFE-specific mapping examples and defaults. If you plan to submit through Slurm, also review SLURM header templates.
Validate the configuration before heavy computation (Optional):
```
batter run examples/rbfe_example.yaml --dry-run
```
This command runs ligand parameterisation (a heavy step) and prepares the equilibration systems. On shared clusters, run the dry-run on a compute node if possible to avoid overloading login nodes.
Inspect the staged system (Optional) Once the dry-run completes, review <run.output_folder>/executions/<run_id>/:
- simulations/<LIGAND>/equil/full.pdb – ligand-specific equilibration systems. Check if the ligand is correctly placed in the binding site, and that membranes/solvent boxes look reasonable.
Launch the full workflow manager (local execution):
```
batter run examples/rbfe_example.yaml
```
Production runs take hours to days depending on system size, the number of ligands, and available hardware. Progress is streamed to the terminal and to executions/<run_id>/logs/batter.log.

Submitting the manager job via SLURM (RECOMMENDED)#

Before submitting, make sure the SLURM header files have been seeded and edited for your cluster. BATTER stores them in ~/.batter/ by default (or under run.slurm_header_dir if you configured a custom location). In particular, update job_manager.header and SLURMM-Am.header so they load Amber/AmberTools successfully and match your site environment (modules, conda activation, partitions, MPI launcher, executable paths, account settings, etc.). If you plan to run REMD, also review SLURMM-BATCH-remd.header. The dedicated SLURM header templates page summarizes what each header controls and how the seeded files relate to the packaged script bodies.

Seed the default headers if needed:

batter seed-headers

To submit the same run through SLURM:

batter run examples/rbfe_example.yaml --slurm-submit

Provide --slurm-manager-path if you maintain a custom SLURM header template (accounts, modules, partitions, etc.). Copy and modify the default template from batter/data/job_manager.header + job_manager.body. See SLURM header templates for the full header layout and override rules.

The job manager stages the system locally, writes an sbatch script based on the YAML hash, and streams updates as windows finish.

Handy CLI Flags#

batter run exposes many overrides so you rarely have to edit YAML mid-iteration:

--on-failure {prune,raise,retry}: Decide how to handle per-ligand failures. retry clears FAILED sentinels and reruns that phase once.
--clean-failures / --no-clean-failures: Remove FAILED sentinels, job_attempt.txt retry counters, and progress caches before rerunning a previous execution.
--only-equil / --full: Stop after shared prep/equilibration—useful for debugging system setup before FE windows.
--dry-run: Stage the system and prepare equilibration inputs without running any MD.
--run-id and --output-folder: Override execution paths without touching system.* fields.
--slurm-submit / --slurm-manager-path: Switch between local execution and SLURM submission (with an optional custom header).

Some failures are transient cluster issues rather than setup problems, for example a job landing on a bad node or hitting a temporary GPU/filesystem problem. In that case, rerun the same command with --clean-failures to clear stale failure markers before resuming. If you want BATTER to clear phase sentinels and retry once within the run manager, use --on-failure retry.

Results and Analysis#

Completed runs automatically write MBAR summaries under results/<run_id>. For RBFE runs, per-run analysis also writes a Cinnabar bundle under results/cinnabar/<run_id>/. The most direct ways to inspect those outputs are:

Open results/cinnabar/<run_id>/cinnabar_dashboard.html in a browser. That dashboard includes the network view, the absolute ranking view, and the clickable ligand / mapping panels.
Read edge_summary.csv when you want the combined edge-level ΔΔG table.
Read cinnabar_relative.csv and cinnabar_absolute.csv when you want the FEMap-exported relative and absolute values.
Open cinnabar_network.png and cinnabar_absolute_sorted.png for static figures suitable for slides or quick sharing.
Use the RBFE Cinnabar analysis notebook when you want notebook-based tables, plots, and optional experimental comparisons.

If you later merge multiple RBFE runs with batter fe cinnabar, the combined bundle is written separately from the per-run subdirectory. Same-work-dir replicates default to results/cinnabar/; cross-work-dir combinations should use an explicit --out-dir.

Use the CLI helpers to inspect them:

batter fe list <run.output_folder>
batter fe show <run.output_folder> <run_id> --ligand <ligand_pair>

For cross-run RBFE benchmarking or Cinnabar plotting, convert stored BATTER records into a Cinnabar bundle. The recommended form treats each run as an atomic WORK_DIR + RUN_ID input, so runs from different work directories can be combined:

batter fe cinnabar \
    --run work/adrb2 rep1 \
    --run work/adrb2_retry rep2 \
    --out-dir combined_cinnabar

Per-run RBFE analysis already writes a default bundle under results/cinnabar/<run_id>/. Use explicit --run inputs when you want to merge replicate runs into one Cinnabar view. If all runs are in the same work directory, this shortcut is equivalent:

batter fe cinnabar <run.output_folder> --run-id rep1 --run-id rep2

The same workflow is available from Python via batter.analysis.cinnabar.build_batter_rbfe_cinnabar_from_runs(). This is the function to use when you want to combine replicate run ids programmatically or connect networks from different work directories. BATTER matches ligand endpoints by ligand name plus canonical SMILES: matching name/SMILES pairs merge into one node, while same-name but different-SMILES endpoints remain separate suffixed nodes.

See Using Cinnabar with RBFE Results for the dedicated Cinnabar workflow page, including the default per-run output layout and the Python API for combined replicate bundles.

Those commands read the saved results/index.csv rows, combine the selected RBFE edges, and write a derived bundle. Use --split-runs only with the same-work-dir shortcut if you want one bundle per run instead of collapsing repeats. If you have experimental absolute affinities, pass them with --experimental-csv so Cinnabar can emit DG/DDG comparison plots. BATTER merges A~B and B~A into one canonical edge by default; add --split-directions if you want to keep the two stored directions separate in the Cinnabar export. BATTER also writes cinnabar_absolute_sorted.png from the Cinnabar MLE absolute values; use --absolute-offset if you want to shift that ranking plot onto a chosen absolute reference level.

fe list prints a high-level table for every stored run, while fe show opens the saved record for one transformation pair such as LIG1~LIG2. For a file-by-file description of the portable repository, including the RBFE-only mapping.*, rbfe_network.png, and Equil_ref / Equil_alt exports, see Results Folder Layout.

For final error estimation, it is usually better to run three independent repeats of the full simulation and estimate the uncertainty across those replicate runs, rather than relying only on the per-run bootstrap uncertainty from a single run. The per-run bootstrapping remains useful as a within-run diagnostic, but it should not be treated as a substitute for repeat-run error estimation.

BATTER does not apply any automatic symmetry correction to the reported free energies. If your transformation needs a symmetry correction, inspect the end states and add that correction separately when interpreting the final result.

Lambda-Schedule Tuning#

If you already know the approximate number of windows your ligand series needs, you can keep that count fixed and use batter fek-schedule to optimize the spacing. The current recipe is documented in Optimizing FEP Schedules from AR Data.

For the small-molecule RBFE cases documented so far, 24 windows often seem to be enough, using a simple evenly spaced schedule:

lambdas: [0.0, 0.04347826, 0.08695652, 0.13043478, 0.17391304,
          0.2173913, 0.26086957, 0.30434783, 0.34782609, 0.39130435,
          0.43478261, 0.47826087, 0.52173913, 0.56521739, 0.60869565,
          0.65217391, 0.69565217, 0.73913043, 0.7826087, 0.82608696,
          0.86956522, 0.91304348, 0.95652174, 1.0]

For more complex transformations, 48 windows has worked well in testing:

lambdas: [0.00000000, 0.12542000, 0.16637000, 0.19653000, 0.22148000, 0.24326000,
          0.26289000, 0.28094000, 0.29779000, 0.31370000, 0.32884000, 0.34336000,
          0.35737000, 0.37095000, 0.38416000, 0.39707000, 0.40971000, 0.42215000,
          0.43441000, 0.44652000, 0.45852000, 0.47043000, 0.48228000, 0.49410000,
          0.50590000, 0.51772000, 0.52958000, 0.54150000, 0.55351000, 0.56563000,
          0.57790000, 0.59036000, 0.60303000, 0.61596000, 0.62920000, 0.64280000,
          0.65684000, 0.67140000, 0.68659000, 0.70254000, 0.71944000, 0.73754000,
          0.75722000, 0.77906000, 0.80408000, 0.83431000, 0.87533000, 1.00000000]