Setting up a DNA-Ligand System for AMBER Simulations parameters subject to change based on system studied
AMBER
Find a PDB of your system.
Download the PDB of the DNA-Ligand into your directory.
Clean up the PDB.
- Ligand PDB -> GCRT
- GCRT -> GESP
- GESP -> mol2/prepi file
- mol2/prepi -> frcmod file
pdb4amber -i orig.pdb -o new.pdb –-reduce –-dry
antechamber -i file.pdb -fi pdb -o file.com -fo gcrt -gv 1 -ge file.gesp -nc 1 (for net charge=1)
run gaussian job -> generate gesp file
antechamber -i file.gesp -fi gesp -o file.mol2 -fo mol2 -c resp -eq 2
antechamber -i file.gesp -fi gesp -o file.prepi -fo prepi -c resp -eq 2
parmchk2 -i file.mol2 -f mol2 -o file.frcmod
make off files now
Creating off (.lib) files
module load amber/ tleap/ source leaprc.gaff2/ MOL=loadmol2 file.mol2/ check MOL/ loadamberparams file.frcmod/ saveoff MOL file.lib/ saveamberparm MOL file.prmtop file.rst7/ quit/
Once completed with the antechamber protocol, the following are the next steps:
###To create your system###
LIGAND ONLY PREP/
source leaprc.gaff2/ source leaprc.water.spce/ loadoff distamycin_B_H.off/ loadamberprep distamycin_B_H.prepi/ loadamberparams distamycin_B_H.frcmod/ MOL = loadpdb distamycin_B_H.pdb/ addions MOL Cl- 0/ solvateoct MOL SPCBOX 12.0/ check MOL/ savepdb MOL final_distamycinB.pdb/ saveamberparm MOL final_distamycinB.prmtop final_distamycinB.inpcrd/ saveamberparm MOL final_distamycinB.prmtop final_distamycinB.rst7/ quit/
LIGAND-DNA COMPLEX PREP 1:1/
source leaprc.DNA.bsc1/ source leaprc. gaff2/ source leaprc. water.spce/ loadoff distamycin_B_H.off/ loadamberprep distamycin_B_H.prepi/ loadamberparams distamycin_B_H.frcmod/ DMY = loadpdb dna_distamycin.pdb/ addions DMY Na+ 0/ solvateoct DMY SPCBOX 12.0/ check DMY/ savepdb DMY final_dna_distamycin.pdb/ saveamberparm DMY final_dna_distamycin.prmtop final_dna_distamycin.inpcrd/ saveamberparm DMY final_dna_distamycin.prmtop final_dna_distamycin.rst7/ quit/
LIGAND-DNA COMPLEX PREP 2:1/
source leaprc.DNA.bsc1/ source leaprc.gaff2/ source leaprc.water.spce/ loadoff distamycin_A_H.off/ loadamberprep distamycin_A_H.prepi/ loadamberparams distamycin_A_H.frcmod/ loadoff distamycin_B_H.off/ loadamberprep distamycin_B_H.prepi/ loadamberparams distamycin_B_H.frcmod/ DMY = loadpdb double_bound_final.pdb/ addions DMY Na+ 0/ solvateoct DMY SPCBOX 12.0/ check DMY/ savepdb DMY final_double_bound.pdb/ saveamberparm DMY final_double_bound.prmtop final_double_bound.inpcrd/ saveamberparm DMY final_double_bound.prmtop final_double_bound.rst7/ quit/
DNA ONLY PREP/
source leaprc.DNA.bsc1/ source leaprc.water.spce/ mol = loadpdb dna_only.pdb/ addions mol Na+ 0/ solvateoct mol SPCBOX 12.0/ savepdb mol dna_only_solv.pdb/ saveamberparm mol dna_only.prmtop dna_only.inpcrd/ saveamberparm mol dna_only.prmtop dna_only.rst7/ check mol / quit/
Once you have created these files (your system), you may proceed with the “New biological molecule (DNA/INC) protocol":
- Initial minimization: Let solvent relax around restrained solute
- 1000 steps (500 steepest descents/500 conjugate gradient)
- 500kcal/mol-Å2 restraints (solute)
- Second minimization: Let everything relax
- 2500 steps (1000 steepest descents/1500 conjugate gradient)
- Defrost (md1): Begin constant volume to warm to proper temperature with restrained solute
- 100ps NPT
- Langevin temperature control 0 -> 300K
- 25 kcal/mol-Å2 restraints (solute)
- Equilibration (md2): Switch to constant pressure to get proper density while gradually releasing restraints on solute (5-stage release with strong restraints for first 40ps while density is changing most rapidly)250ps NPT
- Langevin temperature control 300K
- “Weak-coupling” pressure control 1.0bar (~1atm)
- 25 -> 5kcal/mol-Å2 restraints(solute) o md2a 25kcal 50ps o md2b 20kcal 50ps o md2c 15kcal 50ps o md2d 10kcal 50ps o md2e 5kcal 50ps
- Equilibration (md3): Release solute restraints and collect data to isotropically scale box size to reflect average volume
- 200ps NPT
- Langevin temperature control 300K
- “Weak-coupling” pressure control 1.0bar(~1.0atm)
- calculate new volume and replace x,y,z in restart file
- New flag: ntxo=1
- Equilibration (md4): Switch to constant volume and equilibrate with scaled box size
- 1ns NVT
- Remove ntxo=1 flag
- Production run: keep the same conditions as the equilibration run
- 1 microsecond
- Keep running production for however long is needed for your system until it equilibrates completely.
Input Files for Protocol
Min1.in /
minimization for water and counterions/ &cntrl/ irest=0,/ ntx=1,/ imin=1,/ ntr=1,/ ncyc=500,/ maxcyc=1000,/ ntr=1,/ restraint_wt=500.0,/ restraintmask=':1',/ ##based on what you want to restrain### ntpr=100,/ ntwx=0,/ cut=10.0,/ /
Min2.in /
Solute minimization run/ &cntrl/ imin=1,/ ntx=1,/ irest=0,/ ncyc=1000,/ maxcyc=2500,/ ntpr=100,/ ntwx=0,/ cut=10.0,/ /
Md1.in/
Defrost NVT 0 to 300 K with res on DNA/ &cntrl/ imin=0,/ irest=0,/ ntx=1,/ ntb=1,/ cut=10.0,/ ntr=1,/ restraint_wt=25.0, restraintmask=':1',/ ntc=2,/ ntf=2,/ tempi=0, temp0=300,/ ntt=3, gamma_ln=1.0,/ nstlim=50000, dt=0.002,/ ntpr=100, ntwx=100, ntwr=1000/ /
Md2a.in/
Minimization Restraint 25kcal 50ps/ &cntrl/ imin=0,/ irest=1,/ ntx=5,/ ntb=2,/ pres0=1.0,/ ntp=1,/ taup=1.0,/ cut=10.0,/ ntr=1, restraint_wt=25.0, restraintmask=':1',/ ntc=2, ntf=2,/ tempi=300.0, temp0=300.0,/ ntt=3,/ gamma_ln=1.0,/ nstlim=25000, dt=0.002,/ ntpr=100, ntwx=1000, ntwr=1000/ /
Md2b.in/
Minimization Restraint 20kcal 50ps/ &cntrl/ imin=0,/ irest=1,/ ntx=5,/ ntb=2,/ pres0=1.0,/ ntp=1,/ taup=1.0,/ cut=10.0,/ ntr=1, restraint_wt=20.0, restraintmask=':1',/ ntc=2, ntf=2,/ tempi=300.0, temp0=300.0,/ ntt=3,/ gamma_ln=1.0,/ nstlim=25000, dt=0.002,/ ntpr=100, ntwx=1000, ntwr=1000/ /
Md2c.in/
Minimization Restraint 15kcal 50ps/ &cntrl/ imin=0,/ irest=1,/ ntx=5,/ ntb=2,/ pres0=1.0,/ ntp=1,/ taup=1.0,/ cut=10.0,/ ntr=1, restraint_wt=15.0, restraintmask=':1',/ ntc=2, ntf=2,/ tempi=300.0, temp0=300.0,/ ntt=3,/ gamma_ln=1.0,/ nstlim=25000, dt=0.002,/ ntpr=100, ntwx=1000, ntwr=1000/ /
Md2d.in/
Minimization Restraint 10kcal 50ps/ &cntrl/ imin=0,/ irest=1,/ ntx=5,/ ntb=2,/ pres0=1.0,/ ntp=1,/ taup=1.0,/ cut=10.0,/ ntr=1, restraint_wt=10.0, restraintmask=':1',/ ntc=2, ntf=2,/ tempi=300.0, temp0=300.0,/ ntt=3,/ gamma_ln=1.0,/ nstlim=25000, dt=0.002,/ ntpr=100, ntwx=1000, ntwr=1000/ /
Md2e.in/
Minimization Restraint 5kcal 50ps/ &cntrl/ imin=0,/ irest=1,/ ntx=5,/ ntb=2,/ pres0=1.0,/ ntp=1,/ taup=1.0,/ cut=10.0,/ ntr=1, restraint_wt=5.0, restraintmask=':1',/ ntc=2, ntf=2,/ tempi=300.0, temp0=300.0,/ ntt=3,/ gamma_ln=1.0,/ nstlim=25000, dt=0.002,/ ntpr=100, ntwx=1000, ntwr=1000/ /
Md3.in/
Equilibrization md3/ &cntrl/ imin=0,/ irest=1,/ ntx=5,/ ntb=2,/ pres0=1.0,/ ntp=1,/ taup=1.0,/ cut=10.0,/ ntc=2, ntf=2,/ tempi=300.0, temp0=300.0,/ ntt=3,/ gamma_ln=1.0,/ ntxo=1,/ nstlim=100000, dt=0.002,/ ntpr=100, ntwx=1000, ntwr=1000,/ /
Md4.in/
Equilibrate md4 for 1 ns in NVT/ &cntrl/ imin=0,/ irest=1, ntx=5,/ ntb=1,/ cut=10.0,/ ntc=2, ntf=2,/ tempi=300.0, temp0=300.0,/ ntt=3, gamma_ln=1.0,/ nstlim=500000, dt=0.002,/ ntpr=500, ntwx=1000, ntwr=100000,/ /
Production.in/
Production run NVT/ &cntrl/ imin=0,/ irest=1, ntx=5,/ ntb=1,/ ig=-1,/ cut=10.0,/ ntc=2, ntf=2,/ tempi=300.0, temp0=300.0,/ ntt=3, gamma_ln=1.0,/ nstlim=50000000, dt=0.002,/ ntpr=1000, ntwx=500, ntwr=10000,/ /