gmx-MMPBSA 1.0.0

gmx_MMPBSA is a new tool aid to perform end-state free energycalculations with GROMACS files.

Documentation

Note: We do not intend to replace the original MMPBSA.py; instead, we have implemented and improved some functionalities, and what is most important, made this valuable tool available for Gromacs users. Most of the documentation below is found in the Amber manual, we will point out what is new or different. Neither of these should be considered as a “black-box”, and users should be familiar with Amber and MM/PB(GB)SA method before at-tempting these sorts of calculations. These scripts automate a series of calculations, and cannot trap all the types of errors that might occur. You can review some of the answers to the questions that we consider most common here. If you find a bug or have any question, please consider opening an issue.

Requirements

gmx_MMPBSA requires AmberTools20 to be installed in your machine and the shell environment correctly set up for Amber. The AmberTools suite is free of charge and you can check Amber Manual for a detailed installation guide. Of note, you can have more than one AmberTools installed in your machine. In case AmberTools20 is not the default Amber in your computer, just make sure to source AmberTools20 before installing/updating/running gmx_MMPBSA. gmx_MMPBSA also requires Gromacs (series 4.x.x or 5.x.x or 20xx.x) to be installed in your computer and the shell environment correctly set up for Gromacs. gmx_MMPBSA has been tested with Gromacs4.6.7, 5.1.2 and 2018.3, although it should run smoothly with any Gromacs present in the PATH and that is compatible with the files you are using.

gmx_MMPBSA contains a module that allows for plotting the results. For this, it requires the installation of PyQt5.

amber.python -m pip install PyQt5

Installing gmx_MMPBSA

You can install gmx_MMPBSA from the stable version on PYPI:

amber.python -m pip install gmx_MMPBSA

or the development version from GitHub:

amber.python -m pip install git+https://github.com/Valdes-Tresanco-MS/gmx_MMPBSA

Make sure that you have git installed. If not you can install it as follow:

sudo apt install git

Update

If you already have installed a previous gmx_MMPBSA version, you can update it as follows:

stable version (recommended):

amber.python -m pip install gmx_MMPBSA --upgrade

development version from GitHub:

amber.python -m pip intall git+https://github.com/Valdes-Tresanco-MS/gmx_MMPBSA --upgrade 

Make sure that you have git installed.

We will do our best to keep the PYPI package up to date.

Introduction

Molecular Mechanics / Poisson Boltzmann (or Generalized Born) Surface Area (MM/PB(GB)SA) is a post-processing method in which representative snapshots from an ensemble of conformations are used to calculate the free energy change between two states (typically a bound and free state of a receptor and ligand). Free energy differences are calculated by combining the so-called gas phase energy contributions (MM term) that are independent of the chosen solvent model as well as solvation free energy components (both polar and non-polar) calculated from an implicit solvent model combination (PBSA or GBSA) for each species. Entropy contributions to the total free energy may be added as a further refinement.

The gas phase free energy contributions are calculated by sander or mmpbsa_py_energy within the AmberTools package according to the force field used in the MD simulation. The solvation free energy contributions may be further decomposed into a polar and non-polar contributions. The polar portion is calculated using the Poisson Boltzmann (PB) equation, the Generalized Born method, or the Reference Interaction Site Model (RISM). The PB equation is solved numerically by either the pbsa program included with AmberTools or by the Adaptive Poisson Boltzmann Solver (APBS) program (for more information, see http://www.poissonboltzmann.org/apbs). The non-polar contribution is approximated by the LCPO method implemented within sander or the molsurf method as implemented in cpptraj. The entropy calculations can be done in either a HCT Generalized Born solvation model or in the gas phase using a mmpbsa_py_nabnmode program written in the nab programming language, or via the quasi-harmonic approximation in ptraj as in the original MMPBSA.py. In this new module, entropy term (−TΔS) can be also estimated using the so-called Interaction Entropy method, which is theoretically rigorous, computationally efficient, and numerically reliable for calculating entropic contribution to free energy in protein–ligand binding and other interaction processes.

Usually, the Single Trajectory (ST) approximation is employed when performing MM/PB(GB)SA calculations. This approximation assumes that the configurational space explored by the systems are very similar between the bound and unbound states, so every snapshot for each species (i.e. complex, receptor, and ligand) is extracted from the same trajectory file. This approximation improves binding free energies convergence and also reduces the computing time. However, it only should be applied when the molecules in the unbound state present a similar behavior to that of the bound state. On the other hand, in the so-called Multiple Trajectory (MT) approximation, the snapshots for each one of the species (i.e. complex, receptor, and ligand) are extracted from their own trajectory file. This approximation, theoretically more rigorous though, leads to higher standard deviation of the binding free energies.

Literature

Further information can be found in Amber manual:

• MMPBSA.py
• The Generalized Born/Surface Area Model
• PBSA
• Reference Interaction Site Model
• Generalized Born (GB) for QM/MM calculations

and the foundational papers:

• Srinivasan J. et al., 1998
• Kollman P. A. et al., 2000
• Gohlke H., Case D. A. 2004

as well as some reviews:

• Genheden S., Ryde U. 2015
• Wang et. al., 2018
• Wang et. al., 2019

gmx_MMPBSA in a nutshell

gmx_MMPBSA brings in all the MMPBSA.py functionalities to Gromacs users. In addition, few other functionalities were implemented that eases a number of calculations (e.g. MM/PB(GB)SA with different internal dielectric constant, interaction entropy calculation). A GUI application is also incorporated that allows for visualizing the results and saving high-quality images.

Types of calculations you can do

There are many different options for running gmx_MMPBSA. Among the types of calculations you can do are:

• Normal binding free energies, with either PB or GB implicit solvent models. Each can be done with either 1, 2, or 3 different trajectories, but the complex, receptor, and ligand topology files must all be defined. The complex trajectory must always be provided. Whichever trajectories of the receptor and/or ligand that are NOT specified will be extracted from the complex trajectory. This allows a 1-, 2-, or 3-trajectory analysis. All PB calculations and GB models can be performed with just AmberTools via the mmpbsa_py_energy program installed with MMPBSA.py.
• Stability calculations with any calculation type.
• Alanine scanning with either PB or GB implicit solvent models. All trajectories will be mutated to match the mutated topology files, and whichever calculations that would be carried out for the normal systems are also carried out for the mutated systems. Note that only 1 mutation is allowed per simulation, and it must be to an alanine. If mutant_only is not set to 1, differences resulting from the mutations are calculated. This option is incompatible with intermediate NetCDF trajectories (see the netcdf = 1 option above). This has the same program requirements as option 1 above.
• Entropy corrections. An entropy term can be added to the free energies calculated above using either the quasi-harmonic, the normal mode or interaction entropy approximations. Calculations will be performed for the normal and mutated systems (alanine scanning) as requested. Normal mode calculations are done with the mmpbsa_py_nabnmode program included with AmberTools.
• Decomposition schemes. The energy terms will be decomposed according to the decomposition scheme outlined in the idecomp variable description. This should work with all of the above, though entropy terms cannot be decomposed. APBS energies cannot be decomposed, either. Neither can PBSA surface area terms. This functionality requires sander from the Amber 11 (or later) package.
• QM/MMGBSA. This is a binding free energy (or stability calculation) using the Generalized Born solvent model allowing you to treat part of your system with a quantum mechanical Hamiltonian. See “Advanced Options” for tips about optimizing this option. This functionality requires sander from the Amber package.
• MM/3D-RISM. This is a binding free energy (or stability calculation) using the 3D-RISM solvation model. This functionality is performed with rism3d.snglpnt built with AmberTools.
• Membrane Protein MMPBSA. Calculate the MMPBSA binding free energy for a ligand bound to a protein that is embedded into a membrane. Only use_sander=1 is supported.

Examples...

• Protein-DNA binding free energy calculations
• Protein-ligand binding free energy calculations (Single Trajectory method) (based on this tutorial)
• Protein-ligand binding free energy calculations (Multiple Trajectory method) (based on this tutorial)
• MMPBSA with membrane proteins
• Protein-protein binding free energy calculations (based on this tutorial)
• Protein-protein binding free energy calculations with MM/3D-RISM
• Alanine scanning (based on this tutorial)
• Decomposition analysis (based on this tutorial)
• Entropy calculations with normal modes (based on this tutorial)
• Entropy calculations with Interaction Entropy
• Stability calculations

Calling gmx_MMPBSA from the command-line

gmx_MMPBSA is invoked through the command line as follows:

usage: gmx_MMPBSA [-h] [-v] [--input-file-help] [-O] [-prefix <file prefix>] [-i FILE]
                  [-xvvfile XVVFILE] [-o FILE] [-do FILE] [-eo FILE] [-deo FILE] [-gui] [-s] 
                  [-cs <Structure File>] [-ci <Index File>] [-cg index index] [-ct [TRJ [TRJ ...]]]
                  [-rs <Structure File>] [-ri <Index File>] [-rg index] [-rt [TRJ [TRJ ...]]] 
                  [-lm <Structure File>] [-ls <Structure File>] [-li <Index File>] [-lg index] 
                  [-lt [TRJ [TRJ ...]]] [-make-mdins] [-use-mdins] [-rewrite-output]

gmx_MMPBSA is an effort to bring in Amber's MMPBSA.py functionalities and more to Gromacs users. This 
program is based on Amber's MMPBSA.py and essentially works as such. gmx_MMPBSA minimizes 
compatibility-related issues since it will process any Gromacs files compatible with the Gromacs 
in the path.

optional arguments:
  -h, --help            show this help message and exit
  -v, --version         show program's version number and exit
  --input-file-help     Print all available options in the input file. (default: False)

Miscellaneous Options:
  -O, --overwrite       Allow output files to be overwritten (default: False)
  -prefix <file prefix>
                        Prefix for intermediate files. (default: _GMXMMPBSA_)

Input and Output Files:
  These options specify the input files and optional output files.

  -i FILE               MM/PBSA input file. (default: None)
  -xvvfile XVVFILE      XVV file for 3D-RISM. (default: $AMBERHOME/dat/mmpbsa/spc.xvv)
  -o FILE               Output file with MM/PB(GB)SA statistics. (default: FINAL_RESULTS_MMPBSA.dat)
  -do FILE              Output file for decomposition statistics. (default: FINAL_DECOMP_MMPBSA.dat)
  -eo FILE              CSV-format output of all energy terms for every frame in every calculation.
                         File name forced to end in [.csv]. This file is only written when specified
                         on the command-line. (default: None)
  -deo FILE             CSV-format output of all energy terms for each printed residue in
                         decomposition calculations. 
                         File name forced to end in [.csv]. This file is only written when specified
                         on the command-line. (default: None)
  -gui                  Open GUI plotting app when all calculations are finished (default: True)
  -s                    Perform stability calculation. Only the complex parameters are required. If
                         ligand is non-Protein (small molecule) type, then ligand *.mol2 file is 
                         required. In any other case receptor and ligand parameters will be ignored.
                         See description bellow (default: False)

Complex:
  Complex files and info that are needed to perform the calculation. If the receptor and / or the
    ligand info is not defined, we generate them from that of the complex.

  -cs <Structure File>  Structure file of the complex. If it is Protein-Ligand (small molecule)
                         complex, make sure that you define -lm option. 
                         Allowed formats: *.tpr (recommended), *.pdb, *.gro (default: None)
  -ci <Index File>      Index file of the bound complex. (default: None)
  -cg index index       Groups of receptor and ligand in complex index file. (default: None)
                         Notation: "-cg <Receptor group> <Ligand group>", ie. -cg 1 13
  -ct [TRJ [TRJ ...]]   Input trajectories of the complex. Make sure the trajectory is fitted and
                         pbc have been removed. Allowed formats: *.xtc (recommended), *.trr, *.pdb
                         (specify as many as you'd like). (default: None)

Receptor:
  Receptor files and info that are needed to perform the calculation. If the receptor info is not 
   defined, we generate it from that of the complex.

  -rs <Structure File>  Structure file of the unbound receptor for multiple trajectory approach.
                         Allowed formats: *.tpr (recommended), *.pdb, *.gro (default: None)
  -ri <Index File>      Index file of the unbound receptor. (default: None)
  -rg index             Receptor group in receptor index file. Notation: "-lg <Receptor group>", 
                         e.g. -rg 1 (default: None)
  -rt [TRJ [TRJ ...]]   Input trajectories of the unbound receptor for multiple trajectory approach.
                         Allowed formats: *.xtc (recommended), *.trr, *.pdb, *.gro (specify as many
                         as you'd like). (default: None)

Ligand:
  Ligand files and info that are needed to perform the calculation. If the ligand are not defined, 
   we generate it from that of the complex.

  -lm <Structure File>  A *.mol2 file of the unbound ligand used to parametrize ligand for Gromacs.
                         Must be defined if Protein-Ligand (small molecule) complex was define. 
                         Antechamber output *.mol2 is recommended (default: None)
  -ls <Structure File>  Structure file of the unbound ligand. If ligand is a small molecule, make 
                         sure that you define above -lm option. Allowed formats: *.tpr (recommended),
                         *.pdb, *.gro (default: None)
  -li <Index File>      Index file of the unbound ligand. (default: None)
  -lg index             Ligand group in ligand index file. Notation: "-lg <Ligand group>", 
                         e.g. -lg 13 (default: None)
  -lt [TRJ [TRJ ...]]   Input trajectories of the unbound ligand for multiple trajectory approach. 
                         Allowed formats: *.xtc (recommended), *.trr, *.pdb, *.gro (specify as many
                         as you'd like). (default: None)

Miscellaneous Actions:
  -make-mdins           Create the input files for each calculation and quit. This allows you to 
                         modify them and re-run using -use-mdins (default: False)
  -use-mdins            Use existing input files for each calculation. If they do not exist with the
                         appropriate names, gmx_MMPBSA will quit in error. (default: False)
  -rewrite-output       Do not re-run any calculations, just parse the output files from the 
                         previous calculation and rewrite the output files. (default: False)

This program will calculate binding free energies using end-state free energy methods on an ensemble
of snapshots using a variety of implicit solvent models

Running gmx_MMPBSA

Serial version

This version is installed via pip as described above. AMBERHOME variable must be set, or it will quit with an error. An example command-line call is shown below:

gmx_MMPBSA -O -i mmpbsa.in -cs com.tpr -ci index.ndx -cg 1 13 -ct com_traj.xtc

You can found test files in GitHub

Parallel (MPI) version

Unlike MMPBSA.py, gmx_MMPBSA will be installed as a separate package from the Amber installation. When installing Amber with mpi, a MMPBSA.py version called "MMPBSA.py.MPI" will be installed as well. Since we cannot detect if Amber was installed one way or another, we simply decided to adapt the gmx_MMPBSA executable to use an argument. That is, gmx_MMPBSA is a single script that executes the serial version or the parallel version with mpi depending on whether the user defines the "mpi" or "MPI" argument. In principle, both the serial and parallel versions should work correctly when Amber was installed in parallel.

The parallel version, like MMPBSA.py.MPI requires the mpi4py module. If you did the parallel installation of Amber, it should be installed. In any case, it could be installed in the following way:

amber.python -m pip install mpi4py

A usage example is shown below:

mpirun -np 2 gmx_MMPBSA MPI -O -i mmpbsa.in -cs com.tpr -ci index.ndx -cg 1 13 -ct com_traj.xtc

or

mpirun -np 2 gmx_MMPBSA mpi -O -i mmpbsa.in -cs com.tpr -ci index.ndx -cg 1 13 -ct com_traj.xtc

One note: at a certain level, running RISM in parallel may actually hurt performance, since previous solutions are used as an initial guess for the next frame, hastening convergence. Running in parallel loses this advantage. Also, due to the overhead involved in which each thread is required to load every topology file when calculating energies, parallel scaling will begin to fall off as the number of threads reaches the number of frames.

The input file

As gmx_MMPBSA is based on MMPBSA.py, it uses an input file containing all the specification for the MM/PB(GB)SA calculation. The input file is designed to be as syntactically similar to other programs in Amber as possible. The input file has the same namelist structure as both sander and pmemd. The allowed namelists are &general, &gb, &pb, &rism, &alanine_scanning, &nmode, and &decomp. The input variables recognized in each namelist are described below, but those in &general are typically variables that apply to all aspects of the calculation or parameters required for build amber topologies from Gromacs files. The &gb namelist is unique to Generalized Born calculations, &pb is unique to Poisson Boltzmann calculations, &rism is unique to 3D-RISM calculations, &alanine_scanning is unique to alanine scanning calculations, &nmode is unique to the normal mode calculations used to approximate vibrational entropies, and &decomp is unique to the decomposition scheme. All of the input variables are described below according to their respective namelists. Integers and floating point variables should be typed as-is while strings should be put in either single- or double-quotes. All variables should be set with variable = value and separated by commas. See several examples below. As you will see, several calculations can be performed in the same run (i.e. &gb and &pb, &gb and &alanine_scanning, &pb and &decomp, etc). Variables will usually be matched to the minimum number of characters required to uniquely identify that variable within that namelist. Variables require at least 4 characters to be matched unless that variable name has fewer than 4 characters (in which case the whole variable name is required). For example, “star” in &general will match “startframe”. However, “stare” and “sta” will match nothing.

&general namelist variables

debug_printlevel MMPBSA.py prints errors by raising exceptions, and not catching fatal errors. If debug_printlevel is set to 0, then detailed tracebacks (effectively the call stack showing exactly where in the program the error occurred) is suppressed, so only the error message is printed. If debug_printlevel is set to 1 or higher, all tracebacks are printed, which aids in debugging of issues. Default: 0. (Advanced Option)

startframe The frame from which to begin extracting snapshots from the full, concatenated trajectory comprised of every trajectory file placed on the command-line. This is always the first frame read. (Default = 1)

endframe The frame from which to stop extracting snapshots from the full, concatenated trajectory comprised of every trajectory file supplied on the command-line. (Default = 9999999)

@@ Input variable modified. Included Interaction entropy aproximation @@

entropy It specifies whether to perform a quasi-harmonic entropy (QH) approximation with ptraj or the Interaction Entropy (IE) approximation. The allowed values are (default = 0):

• 0: Don’t
• 1: perform QH
• 2: perform IE

+ New input variable added

entropy_seg Specify the representative segment (in %), starting from the endframe, for the calculation of the Interaction Entropy, e.g.: entropy_seg = 25 means that the last quartile of the total number of frames ((endframe-startframe)/interval) will be used to calculate the average Interaction Entropy. Default: 25 (Only if entropy = 2)

+ New input variable added

entropy_temp Specify the temperature to calculate the entropy term −TΔS (Only if entropy = 2). Avoid inconsistencies with defined internal temperature (298.15 K) when nmode is used (Default = 298.15)

interval The offset from which to choose frames from each trajectory file. For example, an interval of 2 will pull every 2nd frame beginning at startframe and ending less than or equal to endframe. (Default = 1)

- Input variable deleted. All files are needed for plotting

-keep_files The variable that specifies which temporary files are kept. All temporary files have the prefix _GMXMMPBSA_ prepended to them (unless you change the prefix on the command-line—see subsection Subsection 34.3.2 for details). Allowed values are 0, 1, and 2. 0: Keep no temporary files 1: Keep all generated trajectory files and mdout files created by sander simulations 2: Keep all temporary files. Temporary files are only deleted if MMPBSA.py completes successfully (Default = 1) A verbose level of 1 is sufficient to use -rewrite-output and recreate the output file without rerunning any simulations.

netcdf Specifies whether or not to use NetCDF trajectories internally rather than writing temporary ASCII trajectory files. For very large trajectories, this could offer significant speedups, and requires less temporary space. However, this option is incompatible with alanine scanning. Default value is 0.

• 0: Do NOT use temporary NetCDF trajectories
• 1: Use temporary NetCDF trajectories

+ New input variable added

PBRadii PBRadii to build amber topology files (Default = 3):

• 1: bondi, recommended when igb = 7
• 2: mbondi, recommended when igb = 1
• 3: mbondi2, recommended when igb = 2 or 5
• 4: mbondi3, recommended when igb = 8

+ New input variable added

protein_forcefield Define the force field used to build Amber topology for proteins. Make sure this force field is the same as the one used in Gromacs (Default = 3)

• 1: amber99
• 2: amber03
• 3: amber99SB
• 4: amber99SB-ildn
• 5: amber14SB

+ New input variable added

ligand_forcefield Define the force field used to build Amber topology for small molecules. Make sure this force field is the same as the one used in Gromacs (Default = 1). Allowed values are:

• 1: gaff
• 2: gaff2

+ New input variable added

solvated_trajectory Define if it is necessary to build a clean trajectory with no water and ions (Default = 1)

• 0: Don’t
• 1: Build clean trajectory

- Input variable deleted. ALways must be defined to get gromacs

-search_path Advanced option. By default, MMPBSA.py will only search for executables in $AMBERHOME/bin . To enable it to search for binaries in your full PATH if they can’t be found in $AMBERHOME/bin , set search_path to 1. Default 0 (do not search through the PATH ). This is particularly useful if you are using an older version of sander that is not in AMBERHOME .

use_sander use sander for energy calculations, even when mmpbsa_py_energy will suffice (Default = 0)

• 0: Use mmpbsa_py_energy when possible
• 1: Always use sander

verbose The variable that specifies how much output is printed in the output file. There are three allowed values (Default = 1):

• 0: print difference terms
• 1: print all complex, receptor, and ligand terms
• 2: also print bonded terms if one trajectory is used

&gb namelist variables

ifqnt Specifies whether a part of the system is treated with quantum mechanics. This functionality requires sander igb Generalized Born method to use (seeSection 4). Allowed values are 1, 2, 5, 7 and 8. (Default = 5) All models are now available with both mmpbsa_py_energy and sander.(Default = 0)

• 0: Potential function is strictly classical
• 1: Use QM/MM

qm_residues Comma- or semicolon-delimited list of complex residues to treat with quantum mechanics. All whitespace is ignored. All residues treated with quantum mechanics in the complex must be treated with quantum mechanics in the receptor or ligand to obtain meaningful results.

+ Input variable added.

intdiel Define Internal dielectric constant without use external *.mdin file (Default = 1.0)

qm_theory Which semi-empirical Hamiltonian should be used for the quantum calculation. No default, this must be specified. See its description in the QM/MM section of the manual for options.

qmcharge_com The charge of the quantum section for the complex. (Default = 0)

qmcharge_lig The charge of the quantum section of the ligand. (Default = 0)

qmcharge_rec The charge of the quantum section for the receptor. (Default = 0)

qmcut The cutoff for the qm/mm charge interactions. (Default = 9999.0)

saltcon Salt concentration in Molarity. (Default = 0.0)

surfoff Offset to correct (by addition) the value of the non-polar contribution to the solvation free energy term (Default 0.0)

surften Surface tension value (Default = 0.0072). Units in kcal/mol/ Å 2

molsurf When set to 1, use the molsurf algorithm to calculate the surface area for the nonpolar solvation term. When set to 0, use LCPO (Linear Combination of Pairwise Overlaps). (Default 0)

probe Radius of the probe molecule (supposed to be the size of a solvent molecule), in Angstroms, to use when determining the molecular surface (only applicable when molsurf is set to 1). Default is 1.4.

msoffset Offset to apply to the individual atomic radii in the system when calculating the molsurf surface. See the description of the molsurf action command in cpptraj. Default is 0.

&pb namelist variables

inp Option to select different methods to compute non-polar solvation free energy (Default = 2).

• 0: No non-polar solvation free energy is computed
• 1: The total non-polar solvation free energy is modeled as a single term linearly proportional to the solvent accessible surface area, as in the PARSE parameter set, that is, if INP = 1, USE_SAV must be equal to 0.
• 2: The total non-polar solvation free energy is modeled as two terms: the cavity term and the dispersion term. The dispersion term is computed with a surface-based integration method closely related to the PCM solvent for quantum chemical programs. Under this framework, the cavity term is still computed as a term linearly proportional to the molecular solvent-accessible-surface area (SASA) or the molecular volume enclosed by SASA.

cavity_offset Offset value used to correct non-polar free energy contribution (Default = -0.5692) This is not used for APBS.

cavity_surften Surface tension. (Default = 0.0378 kcal/mol Angstrom 2 ). Unit conversion to kJ done automatically for APBS.

exdi External dielectric constant (Default = 80.0).

indi Internal dielectric constant (Default = 1.0).

fillratio The ratio between the longest dimension of the rectangular finite-difference grid and that of the solute (Default = 4.0).

scale Resolution of the Poisson Boltzmann grid. It is equal to the reciprocal of the grid spacing. (Default = 2.0)

istrng Ionic strength in Molarity. It is converted to mM for PBSA and kept as M for APBS. (Default = 0.0)

linit Maximum number of iterations of the linear Poisson Boltzmann equation to try (Default = 1000)

prbrad Solvent probe radius in Angstroms. Allowed values are 1.4 and 1.6 (Default = 1.4)

radiopt The option to set up atomic radii according to 0: the prmtop, or 1: pre-computed values (see Amber manual for more complete description). (Default = 1)

sander_apbs Option to use APBS for PB calculation instead of the built-in PBSA solver. This will work only through the iAPBS interface built into sander.APBS. Instructions for this can be found online at the iAPBS/APBS websites. Allowed values are 0: Don’t use APBS, or 1: Use sander.APBS. (Default = 0)

memopt Turn on membrane protein support (Default = 0).

emem Membrane dielectric constant (Default = 1.0).

mthick Membrane thickness (Default = 40.0).

mctrdz Absolute membrane center in the z-direction (Default=0.0, use protein center as the membrane center).

poretype Turn on the automatic membrane channel/pore finding method (Default=1).

A more thorough description of these and other options can be found here. Please also note that the default options have changed over time. For a detailed discussion of all related options on the quality of the MM/PB(GB)SA calculations, please check this publication.

&alanine_scanning namelist variables

mutant_only Option to perform specified calculations only for the mutants. Allowed values are 0: Do mutant and original or 1: Do mutant only (Default = 0) Note that all calculation details are controlled in the other namelists, though for alanine scanning to be performed, the namelist must be included (blank if desired)

+Two options added to ease the alanine_scanning calculations

mutant Define whether the mutation will be perform in receptor or ligand. Allowed values are: receptor, rec, ligand or lig in any capitalization (Default = receptor or REC)

mutant_res Define the specific residue that is going to be mutated. Use the following format CHAIN:RESNUM (eg: 'A:350'). Please, make sure that your selection is correct and based on Gromacs numbering in processed files.

&nmode namelist variables

dielc Distance-dependent dielectric constant (Default = 1.0)

drms Convergence criteria for minimized energy gradient. (Default = 0.001)

maxcyc Maximum number of minimization cycles to use per snapshot in sander. (Default = 10000)

nminterval ∗ Offset from which to choose frames to perform nmode calculations on (Default = 1)

nmendframe ∗ Frame number to stop performing nmode calculations on (Default = 1000000)

nmode_igb Value for Generalized Born model to be used in calculations. Options are 0: Vacuum, 1: HCT GB model (Default 1)

nmode_istrng Ionic strength to use in nmode calculations. Units are Molarity. Non-zero values are ignored if nmode_igb is 0 above. (Default = 0.0)

nmstartframe ∗ Frame number to begin performing nmode calculations on (Default = 1)

• These variables will choose a subset of the frames chosen from the variables in the &general namelist. Thus, the “trajectory” from which snapshots will be chosen for nmode calculations will be the collection of snapshots upon which the other calculations were performed.

&decomp namelist variables

csv_format Print the decomposition output in a Comma-Separated-Variable (CSV) file. CSV files open natively in most spreadsheets. If set to 1, this variable will cause the data to be written out in a CSV file, and standard error of the mean will be calculated and included for all data. If set to 0, the standard, ASCII format will be used for the output file. Default is 1 (CSV-formatted output file)

dec_verbose Set the level of output to print in the decomp_output file.

• 0: DELTA energy, total contribution only
• 1: DELTA energy, total, sidechain, and backbone contributions
• 2: Complex, Receptor, Ligand, and DELTA energies, total contribution only
• 3: Complex, Receptor, Ligand, and DELTA energies, total, sidechain, and backbone contributions

Note: If the values 0 or 2 are chosen, only the Total contributions are required, so only those will be printed to the mdout files to cut down on the size of the mdout files and the time required to parse them. Default = 0

idecomp Energy decomposition scheme to use:

• 1: Per-residue decomp with 1-4 terms added to internal potential terms
• 2: Per-residue decomp with 1-4 EEL added to EEL and 1-4 VDW added to VDW potential terms.
• 3: Pairwise decomp with 1-4 terms added to internal potential terms
• 4: Pairwise decomp with 1-4 EEL added to EEL and 1-4 VDW added to VDW potential terms

(No default. This must be specified!) This functionality requires sander.

print_res Select residues from the complex to print. Default is print "within 6". This variable also accepts a sequence of individual residues and/or ranges. The different fields must be either comma- or semicolon-delimited. For example: print_res = "within 6", where within corresponds to the keyword and 6 to the maximum distance criterion in Angstroms necessary to select the residues from both the receptor and the ligand; or print_res = “1, 3-10, 15, 100”, or print_res = “1; 3-10; 15; 100”. Both of these will print residues 1, 3 through 10, 15, and 100 from the complex topology file and the corresponding residues in either the ligand and/or receptor topology files.

- *Please note: Using idecomp=3 or 4 (pairwise) with a very large number of printed residues and a
-  large number of frames can quickly create very, very large temporary mdout files. Large print 
-  selections also demand a large amount of memory to parse the mdout files and write 
-  decomposition output file (~500 MB for just 250 residues, since that’s 62500 pairs!) It is not
-  unusual for the output file to take a significant amount of time to print if you have a lot of
-  data. This is most applicable to pairwise decomp, since the amount of data scales as O(N 2 ).

+ Based on the above, we decided to add a new option that limits the selection of the residues 
+ that will be printed by default. We defined a selection method with the following structure:
+ print_res = "within 6" where _within_ corresponds to the keyword and _6_ to the maximum 
+ distance criterion in Angstroms necessary to select the residues from both the receptor and
+ the ligand.

&rism namelist variables*

buffer Minimum distance between solute and edge of solvation box. Specify this with grdspc below. Mutually exclusive with ng and solvbox. Set buffer < 0 if you wish to use ng and solvbox. (Default = 14 Å) closure The approximation to the closure relation. Allowed choices are kh (Kovalenko-Hirata), hnc (Hypernetted- chain), or psen (Partial Series Expansion of order-n) where “n” is a positive integer (e.g., “pse3”). (Default = ‘kh’)

closureorder (Deprecated) The order at which the PSE-n closure is truncated if closure is specified as “pse” or “psen” (no integers). (Default = 1)

grdspc Grid spacing of the solvation box. Specify this with buffer above. Mutually exclusive with ng and solvbox. (Default = 0.5 Å)

ng Number of grid points to use in the x, y, and z directions. Used only if buffer < 0. Mutually exclusive with buffer and grdspc above, and paired with solvbox below. No default, this must be set if buffer < 0. Define like “ng=1000,1000,1000”

polardecomp Decompose the solvation free energy into polar and non-polar contributions. Note that this will increase computation time by roughly 80%. 0: Don’t decompose solvation free energy. 1: Decompose solvation free energy. (Default = 0)

rism_verbose Level of output in temporary RISM output files. May be helpful for debugging or following con- vergence. Allowed values are 0 (just print the final result), 1 (additionally prints the total number of iterations for each solution), and 2 (additionally prints the residual for each iteration and details of the MDIIS solver). (Default = 0)

solvbox Length of the solvation box in the x, y, and z dimensions. Used only if buffer < 0. Mutually exclusive with buffer and grdspc above, and paired with ng above. No default, this must be set if buffer < 0. Define like “solvbox=20,20,20”

solvcut Cutoff used for solute-solvent interactions. The default is the value of buffer. Therefore, if you set buffer < 0 and specify ng and solvbox instead, you must set solvcut to a nonzero value or the program will quit in error. (Default = buffer )

thermo Which thermodynamic equation you want to use to calculate solvation properties. Options are “std”, “gf”, or “both” (case-INsensitive). “std” uses the standard closure relation, “gf” uses the Gaussian Fluctuation approximation, and “both” will print out separate sections for both. (Default = “std”). Note that all data are printed out for each RISM simulation, so no choice is any more computationally demanding than another. Also, you can change this option and use the -rewrite-output flag to obtain a different printout after-the-fact.

tolerance Upper bound of the precision requirement used to determine convergence of the self-consistent solution. This has a strong effect on the cost of 3D-RISM calculations. (Default = 1e-5).

• 3D-RISM calculations are performed with the rism3d.snglpnt program built with AmberTools, written by Tyler Luchko. It is the most expensive, yet most statistical mechanically rigorous solvation model available in MMPBSA.py. See Chapter 7 for a more thorough description of options and theory. A list of references can be found there, too. One advantage of 3D-RISM is that an arbitrary solvent can be chosen; you just need to change the xvvfile specified on the command line (see 34.3.2).

Sample input files

Sample input file for GB and PB calculation
&general
startframe=5, endframe=100, interval=5,
verbose=2, protein_forcefield=3, ligand_forcefield=1,
/
&gb
igb=5, saltcon=0.150,
/
&pb
istrng=0.15, fillratio=4.0
/
--------------------------------------------------------
Sample input file for Alanine scanning
&general
startframe=5, endframe=21, verbose=2, interval=1,
protein_forcefield=3, PBRadii=4
/
&gb
igb=8, saltcon=0.150, intdiel=10
/
&alanine_scanning
#make sure to change this parameter to 'ligand' is the mutation is going to be performed 
#in the ligand
mutant='receptor'
mutant_res='B:65'
/
--------------------------------------------------------
Sample input file for entropy calculations
&general
#
startframe=5, endframe=21, verbose=2, interval=1,
#entropy variable control whether to perform a quasi-harmonic entropy (QH) approximation or the 
#Interaction Entropy (IE)(https://pubs.acs.org/doi/abs/10.1021/jacs.6b02682) approximation
protein_forcefield=3, entropy=2, entropy_seg=25, entropy_temp=298
/
&gb
igb=2, saltcon=0.150,
/
#uncomment the next 4 lines for normal mode calculations
#&nmode
#nmstartframe=5, nmendframe=21, nminterval=2,
#maxcyc=50000, drms=0.0001,
#/
/
--------------------------------------------------------
Sample input file with decomposition analysis
#make sure to include at least one residue from both the receptor
#and ligand in the print_res mask of the &decomp section.
#http://archive.ambermd.org/201308/0075.html
&general
startframe=5, endframe=21, interval=1,
/
&gb
igb=5, saltcon=0.150,
/
&decomp
idecomp=2, dec_verbose=3,
print_res="within 4"
#check _GMXMMPBSA_COM_FIXED.pdb file to select which residues are going to be printed
#in the output file
#print_res="40-41,44,47,78,81-82,85,88,115,118,122,215,218-220,232,241"
/
--------------------------------------------------------
Sample input file for QM/MMGBSA
&general
startframe=5, endframe=100, interval=5,
/
&gb
igb=5, saltcon=0.100, ifqnt=1, qmcharge_com=0,
qm_residues="100-105, 200", qm_theory="PM3"
/
--------------------------------------------------------
Sample input file for MM/3D-RISM
&general
startframe=20, endframe=100, interval=5,
/
&rism
polardecomp=1, thermo="gf"
/
--------------------------------------------------------
Sample input file for MMPBSA with membrane proteins
&general
startframe=1, endframe=100, interval=1, 
debug_printlevel=2, use_sander=1,
/
&pb
radiopt=0, indi=20.0, istrng=0.150,
fillratio=1.25, ipb=1, nfocus=1,
bcopt=10, eneopt=1, cutfd=7.0, cutnb=99.0,
npbverb=1, solvopt=2, inp=2,
memopt=1, emem=7.0, mctrdz=-10.383, mthick=36.086, poretype=1,
maxarcdot=15000
/

A few important notes about input files. Comments are allowed by placing a # at the beginning of the line (whites- pace is ignored). Variable initialization may span multiple lines. In-line comments (i.e., putting a # for a comment after a variable is initialized in the same line) is not allowed and will result in an input error. Variable declarations must be comma-delimited, though all whitespace is ignored. Finally, all lines between namelists are ignored, so comments can be added before each namelist without using #.

The Output File

The header of the output file will contain information about the calculation. It will show a copy of the input file as well as the names of all files that were used in the calculation (topology files and coordinate file(s)). If the masks were not specified, it prints its best guess so that you can verify its accuracy, along with the residue name of the ligand (if it is only a single residue). The energy and entropy contributions are broken up into their components as they are in sander and nmode or ptraj. The contributions are further broken into G gas and G solv . The polar and non-polar contributions are EGB (or EPB) and ESURF (or ECAVITY / ENPOLAR), respectively for GB (or PB) calculations. By default, bonded terms are not printed for any one-trajectory simulation. They are computed and their dif- ferences calculated, however. They are not shown (nor included in the total) unless specifically asked for because they should cancel completely. A single trajectory does not produce any differences between bond lengths, angles, or dihedrals between the complex and receptor/ligand structures. Thus, when subtracted they cancel completely. This includes the BOND, ANGLE, DIHED, and 1-4 interactions. If inconsistencies are found, these values are displayed and inconsistency warnings are printed. When this occurs the results are generally useless. Of course this does not hold for the multiple trajectory protocol, and so all energy components are printed in this case. Finally, all warnings generated during the calculation that do not result in fatal errors are printed after calculation details but before any results.

Temporary Files

gmx_MMPBSA creates working files during the execution of the script beginning with the prefix _GMXMMPBSA_. If gmx_MMPBSA does not finish successfully, several of these files may be helpful in diagnosing the problem. For that reason, every temporary file is described below. Note that not every temporary file is generated in every simulation. At the end of each description, the lowest value of the original “keep_files” variable that will retain this file will be shown in parentheses. Nevertheless, in the current version, all the files are retained for plotting purposes.

make_top.log This file contains the output coming from all the Gromacs programs.

leap.log This file contains the output coming from tleap program.

_GMXMMPBSA_gb.mdin Input file that controls the GB calculation done in sander. (2)

_GMXMMPBSA_pb.mdin Input file that controls the PB calculation done in sander. (2)

_GMXMMPBSA_gb_decomp_com.mdin Input file that controls the GB decomp calculation for the complex done in sander. (2)

_GMXMMPBSA_gb_decomp_rec.mdin Input file that controls the GB decomp calculation for the receptor done in sander. (2)

_GMXMMPBSA_gb_decomp_lig.mdin Input file that controls the GB decomp calculation for the ligand done in sander. (2)

_GMXMMPBSA_pb_decomp_com.mdin Input file that controls the PB decomp calculation for the complex done in sander. (2)

_GMXMMPBSA_pb_decomp_rec.mdin Input file that controls the PB decomp calculation for the receptor done in sander. (2)

_GMXMMPBSA_pb_decomp_lig.mdin Input file that controls the PB decomp calculation for the ligand done in sander. (2)

_GMXMMPBSA_gb_qmmm_com.mdin Input file that controls the GB QM/MM calculation for the complex done in sander. (2)

_GMXMMPBSA_gb_qmmm_rec.mdin Input file that controls the GB QM/MM calculation for the receptor done in sander. (2)

_GMXMMPBSA_gb_qmmm_lig.mdin Input file that controls the GB QM/MM calculation for the ligand done in sander. (2)

_GMXMMPBSA_complex.mdcrd.# Trajectory file(s) that contains only those complex snapshots that will be processed by MMPBSA.py. (1)

_GMXMMPBSA_ligand.mdcrd.# Trajectory file(s) that contains only those ligand snapshots that will be processed by MMPBSA.py. (1)

_GMXMMPBSA_receptor.mdcrd.# Trajectory file(s) that contains only those receptor snapshots that will be processed by MMPBSA.py. (1)

_GMXMMPBSA_complex_nc.# Same as _GMXMMPBSA_complex.mdcrd.#, except in the NetCDF format. (1)

_GMXMMPBSA_receptor_nc.# Same as _GMXMMPBSA_receptor.mdcrd.#, except in the NetCDF format. (1)

_GMXMMPBSA_ligand_nc.# Same as _GMXMMPBSA_ligand.mdcrd.#, except in the NetCDF format. (1)

_GMXMMPBSA_dummycomplex.inpcrd Dummy inpcrd file generated by _GMXMMPBSA_complexinpcrd.in for use with imin=5 functionality in sander. (1)

_GMXMMPBSA_dummyreceptor.inpcrd Same as above, but for the receptor. (1)

_GMXMMPBSA_dummyligand.inpcrd Same as above, but for the ligand. (1)

_GMXMMPBSA_complex.pdb Dummy PDB file of the complex required to set molecule up in nab programs

_GMXMMPBSA_receptor.pdb Dummy PDB file of the receptor required to set molecule up in nab programs

_GMXMMPBSA_ligand.pdb Dummy PDB file of the ligand required to set molecule up in nab programs

_GMXMMPBSA_complex_nm.mdcrd.# Trajectory file(s) for each thread with snapshots used for normal mode calcula- tions on the complex. (1)

_GMXMMPBSA_receptor_nm.mdcrd.# Trajectory file for each thread with snapshots used for normal mode calcula- tions on the receptor. (1)

_GMXMMPBSA_ligand_nm.mdcrd.# Trajectory file for each thread with snapshots used for normal mode calculations on the ligand. (1)

_GMXMMPBSA_ptrajentropy.in Input file that calculates the entropy via the quasi-harmonic approximation. This file is processed by ptraj. (2)

_GMXMMPBSA_avgcomplex.pdb PDB file containing the average positions of all complex conformations processed by

_GMXMMPBSA_cenptraj.in. It is used as the reference for the _GMXMMPBSA_ptrajentropy.in file above. (1)

_GMXMMPBSA_complex_entropy.out File into which the entropy results from _GMXMMPBSA_ptrajentropy.in analysis on the complex are dumped. (1)

_GMXMMPBSA_receptor_entropy.out Same as above, but for the receptor. (1)

_GMXMMPBSA_ligand_entropy.out Same as above, but for the ligand. (1)

_GMXMMPBSA_ptraj_entropy.out Output from running ptraj using _GMXMMPBSA_ptrajentropy.in. (1)

_GMXMMPBSA_complex_gb.mdout.# sander output file containing energy components of all complex snapshots done in GB. (1)

_GMXMMPBSA_receptor_gb.mdout.# sander output file containing energy components of all receptor snapshots done in GB. (1)

_GMXMMPBSA_ligand_gb.mdout.# sander output file containing energy components of all ligand snapshots done in GB. (1)

_GMXMMPBSA_complex_pb.mdout.# sander output file containing energy components of all complex snapshots done in PB. (1)

_GMXMMPBSA_receptor_pb.mdout.# sander output file containing energy components of all receptor snapshots done in PB. (1)

_GMXMMPBSA_ligand_pb.mdout.# sander output file containing energy components of all ligand snapshots done in PB. (1)

_GMXMMPBSA_complex_rism.out.# rism3d.snglpnt output file containing energy components of all complex snap- shots done with 3D-RISM (1)

_GMXMMPBSA_receptor_rism.out.# rism3d.snglpnt output file containing energy components of all receptor snap- shots done with 3D-RISM (1)

_GMXMMPBSA_ligand_rism.out.# rism3d.snglpnt output file containing energy components of all ligand snapshots done with 3D-RISM (1)

_GMXMMPBSA_pbsanderoutput.junk.# File containing the information dumped by sander.APBS to STDOUT. (1)

_GMXMMPBSA_ligand_nm.out.# Output file from mmpbsa_py_nabnmode that contains the entropy data for the ligand for all snapshots. (1)

_GMXMMPBSA_receptor_nm.out.# Output file from mmpbsa_py_nabnmode that contains the entropy data for the receptor for all snapshots. (1)

_GMXMMPBSA_complex_nm.out.# Output file from mmpbsa_py_nabnmode that contains the entropy data for the com- plex for all snapshots. (1)

_GMXMMPBSA_mutant_... These files are analogs of the files that only start with _GMXMMPBSA_ described above, but instead refer to the mutant system of alanine scanning calculations.

_GMXMMPBSA_*out.# These files are thread-specific files. For serial simulations, only #=0 files are created. For parallel, #=0 through NUM_PROC - 1 are created.

Advanced Options

The default values for the various parameters as well as the inclusion of some variables over others in the general MMPBSA.py input file were chosen to cover the majority of all MM/PB(GB)SA calculations that would be attempted while maintaining maximum simplicity. However, there are situations in which MMPBSA.py may appear to be restrictive and ill-equipped to address. Attempts were made to maintain the simplicity described above while easily providing users with the ability to modify most aspects of the calculation easily and without editing the source code.

-make-mdins This flag will create all of the mdin and input files used by sander and nmode so that additional control can be granted to the user beyond the variables detailed in the input file section above. The files created are _GMXMMPBSA_gb.mdin which controls GB calculation; _GMXMMPBSA_pb.mdin which controls the PB calculation; _GMXMMPBSA_sander_nm_min.mdin which controls the sander minimization of snapshots to be prepared for nmode calculations; and _GMXMMPBSA_nmode.in which controls the nmode calculation. If no input file is specified, all files above are created with default values, and _GMXMMPBSA_pb.mdin is created for AmberTools’s pbsa. If you wish to create a file for sander.APBS, you must include an input file with “sander_apbs=1” specified to generate the desired input file. Note that if an input file is specified, only those mdin files pertinent to the calculation described therein will be created!

-use-mdins This flag will prevent MMPBSA.py from creating the input files that control the various calculations (_GMXMMPBSA_gb.mdin, _GMXMMPBSA_pb.mdin, _GMXMMPBSA_sander_nm_min.mdin, and _GMXMMPBSA_nmode.in). It will instead attempt to use existing input files (though they must have those names above!) in their place. In this way, the user has full control over the calculations performed, however care must be taken. The mdin files created by MMPBSA.py have been tested and are (generally) known to be consistent. Modifying certain variables (such as imin=5) may prevent the script from working, so this should only be done with care. It is recommended that users start with the existing mdin files (generated by the -make-mdins flag above), and add and/or modify parameters from there.

-make-mdins and -use-mdins are intended to give added flexibility to user input. If the MM/PBSA input file does not expose a variable you require, you may use the -make-mdins flag to generate the MDIN files and then quit. Then, edit those MDIN files, changing the variables you need to, then running gmx_MMPBSA with -use-mdins to use those modified files.

QM/MMGBSA There are a lot of options for QM/MM calculations in sander, but not all of those options were made available via options in the MMPBSA.py input file. In order to take advantage of these other options, you’ll have to make use of the -make-mdins and -use-mdins flags as detailed above and change the resulting _GMXMMPBSA_gb_qmmm_com/rec/lig.mdin files to fit your desired calculation. Additionally, MMPBSA.py suffers all shortcomings of sander, one of those being that PB and QM/MM are incompatible. Therefore, only QM/MMGBSA is a valid option right now.