- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Just released: The BAT.py 2.4 version, with the following new features:

• Relative binding free energy calculations (RBFE) implemented into the BAT workflow, using both OpenMM and AMBER

• Use of the USalign alignment program instead of lovoalign, with no installation needed

• New and easier instructions to install all the needed dependencies

• Example input files for ranking different ligands

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

See also: GHOAT.py, a fully automated tool for guest-host ABFE calculations using SDR with pmemd.cuda:

https://github.com/GHeinzelmann/GHOAT.py

A tutorial and a detailed user guide are available, as well as necessary parameter and input files for several hosts.

BAT.py v2.4

The Binding Affinity Tool (BAT.py) is a python tool for fully automated absolute binding free energy (ABFE) and relative binding free energy (RBFE) calculations using all-atom Molecular Dynamics (MD). Its workflow encompasses the creation of the bound complex, generation of parameters using Antechamber, preparation of the simulation files, and post-processing to retrieve the binding free energy [1,2]. BAT can set up simulations for the pmemd.cuda software from AMBER, or the OpenMM program combined with OpenMMtools, both capable of performing simulations at a reduced computational cost using graphics processing units (GPUs).

BAT.py can perform ABFE calculations by two alchemical routes in the presence of restraints, either with the double decoupling (DD) procedure or with the simultaneous decoupling and recoupling (SDR) method, the latter suitable for ligands with net charge. For binding free energy calculations using the attach-pull-release (APR) method, download the 1.0 version of the code at the BATv1.0 branch, or the BAT 1.0 release.

The RBFE calculations for BAT.py were just released and are still in their beta version, but they are already fully operational. To know more about the theory and how to perform them, please read the RBFE section at the end of the ABFE tutorial, and also the Relative User Guide available inside the ./doc/ folder.

In addition to AMBER pmemd.cuda or OpenMM, BAT.py also requires a few additional programs to work properly, which are listed in the next section.

Getting started

To use BAT.py, download the files from this repository, which already contain an example for ligand binding to the second bromodomain of the BRD4 protein - BRD4(2). In order to perform all the steps from BAT.py, the following programs must be installed and in your path:

VMD (Visual Molecular Dynamics) [3] - https://www.ks.uiuc.edu/Development/Download/download.cgi?PackageName=VMD

Openbabel 2.4.1 [4] - https://github.com/openbabel/openbabel/releases/tag/openbabel-2-4-1

USalign: Universal structural alignment [5] - https://zhanggroup.org/US-align/ (No installation needed.)

AmberTools20 or later [6] - http://ambermd.org/AmberTools.php

pmemd.cuda software from AMBER (version 20 or later) [7] - http://ambermd.org/GetAmber.php ^a

^a Not needed if using OpenMM for the simulations

A quick installation guide for all the dependencies, using the Anaconda package manager, is provided in the Quick-installation-tutorial.pdf file, located inside the ./doc folder. This file also provides a short and command-oriented tutorial for running the BAT program with OpenMM/OpenMMtools.

The folder ./BAT/all-poses contains examples of input coordinate files, with a docked receptor from the 5uez crystal structure (LMCSS-5uf0_5uez_docked.pdb), as well as 9 docked poses for the ligand with the 5uf0 crystal structure (pose0.pdb to pose8.pdb). The docking files were generated and converted to .pdb using Autodock Vina and AutodockTools, following a protocol adapted from the CELPP challenge tutorial (https://docs.google.com/document/d/1iJcPUktbdrRftAA8cuVa32Ri1TPr2XvZVqTccDja2OM). Inside the ./all-poses folder there is also the original crystal structure file for 5uf0, in pdb format. Below we show an example of using these files to calculate the standard binding free energies of the top 5 docked poses and the crystal structure, with all the necessary steps in the calculation.

Running a sample calculation with AMBER

The simulations and analysis from this tutorial will be performed inside the ./BAT folder. The simulations are divided in two steps, equilibration and free energy calculation. We will use the BAT.py input file called input-dd-amber.in, which has the needed parameters to perform full double decoupling calculations with restraints to five docked poses. Other input files examples using SDR, merged restraints and other options, can be found inside the example-input-files folder.

Briefly, the poses_list parameter in the BAT input file sets up the calculation for the first 5 poses from Autodock Vina, and the celpp_receptor parameter defines the name of the receptor. The file can be modified to perform the calculations in the 5uf0 crystal structure instead, by changing the calc_type option to "crystal", the celpp_receptor option to "5uf0", and the ligand_name option to "89J", which is the ligand residue name in the 5uf0 pdb structure. More details on the various BAT parameters can be found in the user guide, located inside the ./doc folder.

Equilibration

The equilibration step starts from the docked complex or the crystal structure, gradually releasing restraints applied on the ligand and then performing a final simulation with an unrestrained ligand. The necessary simulation parameters for the ligand are also generated in this stage, using the General Amber Force Field versions 1 and 2 (GAFF or GAFF2) [6], and the AM1-BCC charge model [8,9]. To run this step, inside the program main folder type:

python BAT.py -i input-dd-amber.in -s equil

BAT.py is compatible with python 3.8 versions. If you have another version, or you find that this command gives an error, you can use the python version included in the Ambertools20 distribution:

$AMBERHOME/miniconda/bin/python BAT.py -i input-dd-amber.in -s equil

This command will create an ./equil folder, with one folder inside for each of the docked poses (pose0, pose1, etc.). In order to run the simulations for each pose, you can use the run-local.bash script (to run them locally), or the provided PBS-run or SLURMM-run scripts, which are designed to run in a queue system such as TORQUE. Both of these files might have to be adjusted, depending on your computer or server configuration, which can be done in the templates located in the ./BAT/run_files folder. The number of simulations and the applied restraints will depend on the release_eq array defined in the input file.

Free energy calculation

Simulations

Once the equilibration simulations for all poses are finished, the user will now perform the free energy stage. Here, starting from the final state of the equilibrated system, BAT will reset the ligand anchor atoms and the restraints reference values for use in the free energy calculation. In this example we will use the DD method with restraints to obtain the binding free energies. For charged ligands, one should use the SDR method instead, in order to avoid artifacts arising from the periodicity of the system [1]. Again in the program main folder, type:

python BAT.py -i input-dd-amber.in -s fe

For each pose or crystal structure, a folder will be created inside ./fe, and inside there will be two folders, ./rest and ./dd. The restraints (rest) folder contains all the simulations needed for the application/removal of restraints. The ./dd folder contains the coupling/decoupling of the ligand electrostatic/LJ interactions, both in the binding site and in bulk. A script called run-all-dd.bash, inside the ./run_files folder, can be used to run these simulatons quickly using the SLURMM scripts provided. A similar script can be written to do the same, using your particular running protocol.

Analysis

Once all of the simulations are concluded, it is time to process the output files and obtain the binding free energies. Here we use a few parameters already set in the input file, such as using TI or MBAR [10] for the decoupling/recoupling components, and the number of data blocks used to calculate the uncertainties. Inside the main folder type:

python BAT.py -i input-dd-amber.in -s analysis

You should see a ./Results directory inside each ./fe/pose folder, containing all the components and the final calculated binding free energy, located in the Results.dat file. This folder also contains the results for each of the chosen data blocks, used to calculate the uncertainties, as well as the equilibrated structure of the protein-ligand complex used as the restraints reference state. This fully automated procedure can be readily applied for any other ligand that binds to the second BRD4 bromodomain, and with minimal adjustments it can be extended to several other proteins.

Using the SDR method and merged restraints

The SDR method is suitable for ligands with net charge, since it keeps the two MD topologies with the same charge during the transformations. To apply SDR, a few parameters have to be changed or added, which is shown in the example-input-files/input-sdr-amber.in file. This input file also uses the merged components for the restraints, which are explained in detail in the User Guide and in Ref [1].

To apply the merged SDR method, all that is needed is to perform the free energy and analysis steps again using the input-sdr-amber.in file. The full DD and merged SDR methods should produce consistent results if the reference state is the same from the equilibrium stage.

The user can also mix and match the separated/merged restraint components and the DD/SDR methods, for example using double decoupling with the merged restraints or the SDR method with the separated restraint components. Another possibility is to merge only the releasing or the attaching restraints, while keeping the others separate.

Computational cost

The full ABFE calculation for a single pose requires a total of 148.0 nanoseconds of simulations for the DD method, and 100.8 ns for the merged SDR method, which can be achieved in less than one day using a single GTX 1070 NVIDIA GPU. This time is significantly reduced when using more modern GPUs, such as the NVIDIA RTX 20 and RTX 30 series. Furthermore, the free energy simulations from BAT are separated into several independent windows, which allows for trivial parallelization across multiple GPUs.

Ref [1] shows how the simulation times of ABFE calculations using BAT can be reduced even more, to as little as 20 ns per calculation, and still produce accurate results. The files needed to reproduce the paper results were to published with the original paper, but were made available in a subsequent correction:

https://pubs.acs.org/doi/10.1021/acs.jctc.4c01153

The user should download the input-files.zip file from the link above. The README file included explains how to use these input files with the latest BAT2 distribution.

Performing the calculations with OpenMM

BAT also allows the user to run all simulations using the free OpenMM engine [11-15] with OpenMMTools [16]. In this case, instead of the pmemd.cuda software from AMBER, OpenMM 7.7.0 or later and OpenMMTools 0.21.3 or later should be installed and in your path. See the Quick-installation-tutorial.pdf file, located inside the ./doc folder, for instructions on how to install these two programs.

The OpenMM simulations are fully integrated into the BAT workflow, with the equilibration, free energy and analysis steps performed the same way as explained in the tutorial above. Example input files are provided for the OpenMM software as well: input-sdr-openmm.in for the merged SDR method, and example-input-files/input-dd-openmm.in for double decoupling with separated restraints. The simulation time per calculation for each approach is also 100.8 ns and 148 ns, respectively. More details on the OpenMM-specific BAT input variables can be found in the User Guide.

In order to run the equilibration and free energy simulations in the respective folders, inside them are included a bash script, to perform the simulations in a local machine, as well as the PBS-run and SLURMM-run scripts. Both of these files might have to be adjusted, depending on your computer or server configuration. After concluding the simulations and performing the final analysis step, the results will be written in the same location and in the same format as with the AMBER version.

The user might want to compare the AMBER and OpenMM free energy results calculated over the same equilibrated poses states. In that case, the equilibration step should be performed using AMBER, and the free energy step can be performed using both AMBER and OpenMM. Performing equilibration with the latter and free energy with the former may cause problems due to incompatibility between input/output files.

Extending it to other systems

Additional ligands to BRD4(2)

The sample system shown here uses a particular ligand that binds to the second bromodomain of the BRD4 protein - BRD4(2). The system alignment, parameter generation and assignment of the ligand anchor atoms is done automatically, so these same calculations can be extended to any other ligand that binds to this receptor. The only thing needed is the files in the ./all-poses folder to be changed, including the docked receptor and poses pdb files, as well as the crystal structure if desired.

Additional receptors

To include a new receptor system, some additional input data is needed. They include a reference.pdb file to align the system using USalign, three chosen protein anchors, and possibly a few variables for ligand anchor atom search. These can be found inside the ./systems-library folder for three other bromodomains (CREBBP, BRD4(1) and BAZ2B) and the T4 Lysozyme. Other systems will be added with time, as the program is further tested and validated.

RBFE calculations

The relative calculations from BAT.py will use the separate topologies (SepTop) approach, which was proposed originally by Rocklin et al. [17], and recently reintroduced by Baumann et al. [18]. Here we propose a slightly different cycle, which makes our calculations also suitable for transformations between ligands that have different net charges.

A few example input files for RBFE calculations are provided inside the ./BAT/example-input-files/ folder, which can be used the same way as explained in the ABFE tutorial above. More details on the meaning of each file are provided in the Relative User Guide included inside the ./doc folder.

More information and BAT.py citations

The complete BAT.py theoretical background and calculation procedures are available in Refs. [1,2]. The OpenMM calculations are based on David Huggins work on ABFE calculations, available at Ref [19]. Please cite these references if using the BAT code. For more information you can contact the author (me) directly:

Germano Heinzelmann
Departamento de Física, Universidade Federal de Santa Catarina
Florianópolis - SC 88040-970 Brasil
email: [email protected]

I provide free support (to an extent) for academic institutions, or in specific cases in which there might be a bug in the code.

Acknowledgments

Germano Heinzelmann thanks FAPESC and CNPq for the research grants, also Michael Gilson and David Huggins for the support on developing the code.

References

1. G. Heinzelmann, D. J. Huggins and M. K. Gilson (2024). “BAT2: an Open-Source Tool for Flexible, Automated, and Low Cost Absolute Binding Free Energy Calculations”. Journal of Chemical Theory and Computation, 20, 6518.

2. G. Heinzelmann and M. K. Gilson (2021). “Automation of absolute protein-ligand binding free energy calculations for docking refinement and compound evaluation”. Scientific Reports, 11, 1116.

3. W. Humphrey, A. Dalke and K. Schulten. (1996) "VMD - Visual Molecular Dynamics", Journal of Molecular Graphics, 14, 33-38.

4. N. M. O'Boyle, M. Banck, C. A. James, C. Morley, T. Vandermeersch, and G. R. HutchisonEmail. (2011) "Open Babel: An open chemical toolbox." Journal of Cheminformatics, 3, 33.

5. C. Zhang, M. Shine, A. M. Pyle, and Y. Zhang (2022) "US-align: Universal Structure Alignment of Proteins, Nucleic Acids and Macromolecular Complexes." Nature Methods, 19, 1109-1115.

6. J. Wang, R.M. Wolf, J.W. Caldwell, and P. A. Kollman, D. A. Case (2004) "Development and testing of a general AMBER force field". Journal of Computational Chemistry, 25, 1157-1174.

7. D.A. Case, K. Belfon, I.Y. Ben-Shalom, S.R. Brozell, D.S. Cerutti, T.E. Cheatham, III, V.W.D. Cruzeiro, T.A. Darden, R.E. Duke, G. Giambasu, M.K. Gilson, H. Gohlke, A.W. Goetz, R. Harris, S. Izadi, S.A. Izmailov, K. Kasavajhala, A. Kovalenko, R. Krasny, T. Kurtzman, T.S. Lee, S. LeGrand, P. Li, C. Lin, J. Liu, T. Luchko, R. Luo, V. Man, K.M. Merz, Y. Miao, O. Mikhailovskii, G. Monard, H. Nguyen, A. Onufriev, F.Pan, S. Pantano, R. Qi, D.R. Roe, A. Roitberg, C. Sagui, S. Schott-Verdugo, J. Shen, C. Simmerling, N.R.Skrynnikov, J. Smith, J. Swails, R.C. Walker, J. Wang, L. Wilson, R.M. Wolf, X. Wu, Y. Xiong, Y. Xue, D.M. York and P.A. Kollman (2020), AMBER 2020, University of California, San Francisco.

8. A. Jakalian, B. L. Bush, D. B. Jack, and C.I. Bayly (2000) "Fast, efficient generation of high‐quality atomic charges. AM1‐BCC model: I. Method". Journal of Computational Chemistry, 21, 132-146.

9. A. Jakalian, D. B. Jack, and C.I. Bayly (2002) "Fast, efficient generation of high‐quality atomic charges. AM1‐BCC model: II. Parameterization and validation". Journal of Computational Chemistry, 16, 1623-1641.

10. M. R. Shirts and J. Chodera (2008) “Statistically optimal analysis of samples from multiple equilibrium states.” Journal of Chemical Physics, 129, 129105.

11. M. S. Friedrichs, P. Eastman , V. Vaidyanathan, M. Houston, S. LeGrand, A. L. Beberg, D. L. Ensign, C. M. Bruns, and V. S. Pande (2009). "Accelerating molecular dynamic simulations on graphics processing unit." Journal of Computational Chemistry, 30, 864.

12. P. Eastman and V. S. Pande (2010). "OpenMM: A hardware-independent framework for molecular simulations." Computing in science and engineering, 12, 34.

13. P. Eastman and V. S. Pande (2010). "Efficient nonbonded interactions for molecular dynamics on a graphics processing unit." Journal of Computational Chemistry, 31, 1268.

14. P. Eastman and V. S. Pande (2010). "Constant constraint matrix approximation: A robust, parallelizable constraint method for molecular simulations." Journal of Chemical Theory and Computation, 6, 434.

15. P. Eastman, J. Swails, J. D. Chodera, R. T. McGibbon, Y. Zhao, K. A. Beauchamp, L.-P. Wang, A. C. Simmonett, M. P. Harrigan, C. D. Stern, R. P. Wiewiora, B. R. Brooks, and V. S. Pande (2017). “OpenMM 7: Rapid development of high performance algorithms for molecular dynamics.” PLOS Computational Biology, 13, e1005659.

16. J. D. Chodera and M. R. Shirts (2011). "Replica exchange and expanded ensemble simulations as Gibbs multistate: Simple improvements for enhanced mixing." Journal of Chemical Physics, 135, 194110.

17. G. J. Rocklin, D. L. Mobley and K. A. Dill (2023). “Separated topologies: A method for relative binding free energy calculations using orientational restraints”. Journal of Chemical Physics, 138, 085104.

18. H. M. Baumann, E. Dybeck, C. L. McClendon, F. C. Pickard IV, V. Gapsys, L. Pérez-Benito, D. F. Hahn, G. Tresadern, A. M. Mathiowetz and D. L. Mobley (2023) “Broadening the Scope of Binding Free Energy Calculations Using a Separated Topologies Approach”. Journal of Chemical Theory and Computation, 19, 5058.

19. D. J. Huggins (2022) "Comparing the Performance of Different AMBER Protein Forcefields, Partial Charge Assignments, and Water Models for Absolute Binding Free Energy Calculations." Journal of Chemical Theory and Computation, 18, 2616.