(Note: These tutorials are meant to provide illustrative examples of how to use the AMBER software suite to carry out simulations that can be run on a simple workstation in a reasonable period of time. They do not necessarily provide the optimal choice of parameters or methods for the particular application area.)
Copyright McGee, Miller, and Swails 2009

AMBER ADVANCED TUTORIALS
TUTORIAL 3 - SECTION 3.1

Python Script MMPBSA.py

Dwight McGee, Bill Miller III, and Jason Swails

The important files for calculating the binding free energy using MMPBSA.py are the topology files and the mdcrd file (ras-raf_top_mdcrd.tgz)

Calculate the binding free energy of Ras-Raf.

We will now calculate the interaction energy and solvation free energy for the complex, receptor and ligand and average the results to obtain an estimate of the binding free energy. Please note that we will not perform a calculation of the entropy contribution to binding in this part of the tutorial and so strictly speaking our result will not be a true free energy but could be used to compare against similar systems. See Section 3.5 for an example of using Normal Mode Analysis (Nmode) to calculate the entropy contribution for a system or uncomment out the last line in the &general namelist of the input file below to perform a Quasi-Harmonic entropy calculation using the ptraj module in AMBER.

We will carry out the binding energy calculation using both the MM-GBSA method and the MM-PBSA method for comparison. This is accomplished with the following input file for MMPBSA.py:

mmpbsa.in
Input file for running PB and GB
&general
   endframe=50, verbose=1,
#   entropy=1,
/
&gb
  igb=2, saltcon=0.100
/
&pb
  istrng=0.100,
/

The input files for MMPBSA.py are designed to be similar to the setup of an mdin file used in the sander module of AMBER. The start of each namelist is designated by an ampersand (&) followed by the name of the namelist. Furthermore, a backslash (/) or '&end' can be used to end the namelist. For a complete list of all variables please see the User's Manual here. This input file is divided into three namelists: general, pb, and gb. The general namelist is designed to specify variables that are not specific to a particular part of the calculation, but to all parts. In this setup we have defined RAS to be the receptor and RAF to be the ligand. The 'endframe' variable sets what frame of the mdcrd to stop on. The '&gb' and '&pb' namelist markers let the script know to perform MM-GBSA and MM-PBSA calculations with the given values defined within those namelists. The 'verbose' variable allows the user to specify how much output is written to the output file.

The four python scripts (MMPBSA.py, utils.py, alamdcrd.py, and inputparse.py) should have been placed in $AMBERHOME/bin/ during installation. The script can be initiated (using the above input file) using analagous command-line flags to those used by sander and pmemd.

$AMBERHOME/bin/MMPBSA.py -O -i mmpbsa.in -o FINAL_RESULTS_MMPBSA.dat -sp ras-raf_solvated.prmtop -cp ras-raf.prmtop -rp ras.prmtop -lp raf.prmtop -y *.mdcrd

This will run the script interactively and print the progress of the calculation to STDOUT and any errors or warnings to STDERR. Finally, timings will be printed once the calculation has completed showing the time taken during each step of the calculation.

Command-line arguments can be given with shell-recognized wildcards (i.e. * and ? for bash). For example, the '-y *.mdcrd' on the command line tells the script to read in all files in the working directory that end in '.mdcrd' and use them as the trajectories to be analyzed.

Here are all the output files created by this script: pb_gb_output1.tgz.

The script creates three unsolvated mdcrd files (complex, receptor, and ligand) using ptraj that are the coordinates analyzed during the GB and PB calculations. The *.mdout files contain the energies for all frames specified. A PDB file of the average structure is created align (via RMS) all snapshots to prepare for a quasi-harmonic entropy calculation with ptraj if one is requested. All files created by MMPBSA.py should begin with the prefix '_MMPBSA_' except for the final output file, FINAL_RESULTS_MMPBSA.dat.

FINAL_RESULTS_MMPBSA.dat
| Run on Thu Feb 11 12:18:37 EST 2010

|Input file:
|--------------------------------------------------------------
|Input file for running PB and GB
|&general
|   endframe=50, verbose=1,
|#   entropy=1,
|/
|&gb
|  igb=2, saltcon=0.100
|/
|&pb
|  istrng=0.100, 
|/
|--------------------------------------------------------------
|Solvated complex topology file:  ras-raf_solvated.prmtop
|Complex topology file:           ras-raf.prmtop
|Receptor topology file:          ras.prmtop
|Ligand topology file:            raf.prmtop
|Initial mdcrd(s):                prod.mdcrd
|
|Best guess for receptor mask:   ":1-166"
|Best guess for  ligand  mask:   ":167-242"

|Calculations performed using 50 frames.
|Poisson Boltzmann calculations performed using internal PBSA solver in sander.
|
|All units are reported in kcal/mole.
-------------------------------------------------------------------------------
-------------------------------------------------------------------------------

GENERALIZED BORN:

Complex:
Energy Component            Average              Std. Dev.   Std. Err. of Mean
-------------------------------------------------------------------------------
VDWAALS                  -1863.7944               17.1704              2.4283
EEL                     -17200.7297               75.9366             10.7391
EGB                      -3249.6511               65.2075              9.2217
ESURF                       91.3565                1.3938              0.1971

G gas                   -19064.5240               77.8536             11.0102
G solv                   -3158.2946               65.2224              9.2238

TOTAL                   -22222.8186               51.0216              7.2155


Receptor:
Energy Component            Average              Std. Dev.   Std. Err. of Mean
-------------------------------------------------------------------------------
VDWAALS                  -1268.1888               14.2342              2.0130
EEL                     -11557.0773               71.7127             10.1417
EGB                      -2532.0669               57.7003              8.1600
ESURF                       64.2843                1.1143              0.1576


G gas                   -12825.2661               73.1118             10.3396
G solv                   -2467.7826               57.7110              8.1616

TOTAL                   -15293.0487               35.3527              4.9996


Ligand:
Energy Component            Average              Std. Dev.   Std. Err. of Mean
-------------------------------------------------------------------------------
VDWAALS                   -529.3090                9.4198              1.3322
EEL                      -4684.4720               36.1449              5.1117
EGB                      -1688.9631               26.5353              3.7527
ESURF                       37.0493                0.6185              0.0875


G gas                    -5213.7811               37.3522              5.2824
G solv                   -1651.9138               26.5425              3.7537

TOTAL                    -6865.6949               25.8878              3.6611


Differences (Complex - Receptor - Ligand):
Energy Component            Average              Std. Dev.   Std. Err. of Mean
-------------------------------------------------------------------------------
VDWAALS                    -66.2966                4.2751              0.6046
EEL                       -959.1803               34.9190              4.9383
EGB                        971.3789               33.0497              4.6739
ESURF                       -9.9770                0.3759              0.0532


DELTA G gas              -1025.4769               35.1797              4.9752
DELTA G solv               961.4018               33.0518              4.6742


 DELTA G binding =        -64.0750     +/-      6.3729                 0.9013
-------------------------------------------------------------------------------
-------------------------------------------------------------------------------

POISSON BOLTZMANN:

Complex:
Energy Component            Average              Std. Dev.   Std. Err. of Mean
-------------------------------------------------------------------------------
VDWAALS                  -1863.7944               17.1704              2.4283
EEL                     -17200.7297               75.9366             10.7391
EPB                      -3207.7160               66.4023              9.3907
ECAVITY                     67.8762                0.7818              0.1106

G gas                   -19064.5240             6061.1875            857.1813
G solv                   -3139.8399               66.4069              9.3914

TOTAL                    -7686.8660               52.5400              7.4303


Receptor:
Energy Component            Average              Std. Dev.   Std. Err. of Mean
-------------------------------------------------------------------------------
VDWAALS                  -1268.1888               14.2342              2.0130
EEL                     -11557.0773               71.7127             10.1417
EPB                      -2483.7242               56.4551              7.9840
ECAVITY                     47.1495                0.4737              0.0670

G gas                   -12825.2661             5345.3320            755.9441
G solv                   -2436.5747               56.4571              7.9842

TOTAL                    -5250.2060               38.5188              5.4474


Ligand:
Energy Component            Average              Std. Dev.   Std. Err. of Mean
-------------------------------------------------------------------------------
VDWAALS                   -529.3090                9.4198              1.3322
EEL                      -4684.4720               36.1449              5.1117
EPB                      -1670.4169               27.6694              3.9131
ECAVITY                     28.0328                0.4133              0.0584

G gas                    -5213.7811             1395.1865            197.3092
G solv                   -1642.3841               27.6725              3.9135

TOTAL                    -2350.3020               25.1197              3.5525


Differences (Complex - Receptor - Ligand):
Energy Component            Average              Std. Dev.   Std. Err. of Mean
-------------------------------------------------------------------------------
VDWAALS                    -66.2966                4.2751              0.6046
EEL                       -959.1803               34.9190              4.9383
EPB                        946.4251               34.5128              4.8808
ECAVITY                     -7.3062                0.3004              0.0425

DELTA G gas              -1025.4769             1237.6138            175.0250
DELTA G solv               939.1189               34.5141              4.8810



 DELTA G binding =        -86.3579     +/-      8.3264                 1.1775
-------------------------------------------------------------------------------
-------------------------------------------------------------------------------

WARNINGS:
igb=2 should be used with mbondi2 pbradii set. Yours are modified Bondi radii (mbondi)

The beginning of the statistics file includes the date/time, any warnings based on the values and files given, the mmpbsa.in text, the files used by the script, the number of frames analyzed, and which PB solver (if any) was used. The rest of the statistics file includes all the average energies, standard deviations, and standard error of the mean for GB followed by PB. After each section, the ΔG of binding is given along with the error values. The meaning of the different terms in this file is as follows:

VDWAALS = van der Waals contribution from MM.
EEL = electrostatic energy as calculated by the MM force field.
EPB/EGB = the electrostatic contribution to the solvation free energy calculated by PB or GB respectively.
ECAVITY = nonpolar contribution to the solvation free energy calculated by an empirical model.
DELTA G binding = final estimated binding free energy calculated from the terms above. (kCal/mol)

Note that the total gas phase energy has not been reported because the values of the bonded potential terms for the receptor and ligand should exactly cancel those for the complex using the single trajectory approach. An error message will result if the energies do not cancel within an allowed tolerance.

One would typically expect to find an extremely favorable electrostatic energy and a unfavorable solvation free energy. This symbolises the energy that one has to use to de-solvate the binding particles and to align their binding interfaces.

From the negative total binding free energy -86.36 kcal/mol we clearly see that this is a favorable protein-protein complex in pure water but keep in mind that the result does not equal the real binding free energy since we did not estimate the (unfavorable) entropy contribution to binding. Note that the GB approach gives a slightly lower binding energy but still suggests that this is a favorable bound state.


CLICK HERE TO GO TO SECTION 3.2

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(Note: These tutorials are meant to provide illustrative examples of how to use the AMBER software suite to carry out simulations that can be run on a simple workstation in a reasonable period of time. They do not necessarily provide the optimal choice of parameters or methods for the particular application area.)
Copyright McGee, Miller, and Swails 2009