Dear Amber Users
I am trying to optimize my protein structures obtained from
CYANA but I have several doubts about the scripts I wrote to perform
NMR refinement of structures in explicit solvent using AMBER 9.
I am using one of the protocols described by Xia et. al.
(Journal of Biomolecular NMR, 22: 317 – 311, 2002) :
My question is about the parameters I entered in the scripts to
follow the protocol, because in the paper there are some parameters
that were not described and I do not know if I am entering good
values for my requirements.
The protocol is:
REFINEMENT IN VACUUM
1000 steps energy minimization without any constraints
&cntrl
ntwe = 5, ntwx = 5, ntpr = 5,
scnb = 2.0, scee = 1.2, nsnb = 25, dielc = 1, cut = 15.0,
ntb = 0, igb = 0,
maxcyc = 1000, ntmin = 1, ncyc = 100, dx0 = 0.01, drms = 0.01,
ntr = 0,
imin = 1, nmropt = 0,
/
then 2 cycles of 30 ps MD simulated annealing in vacuum, from
1000 K to 0 K
&cntrl
nstlim=30000, pencut=-0.001,
nmropt=1,
ntpr=200, ntt=1, ntwx=200,
cut=15.0, ntb=0, vlimit=10,
/
&ewald
eedmeth=5,
/
&wt type='TEMP0',
istep1=0,istep2=30000,value1=1000.,
value2=0.0, /
&wt type='TAUTP',
istep1=0,istep2=30000,value1=1.0,
value2=1.0, /
&wt type='REST',
istep1=0,istep2=30000,value1=1.0,
value2=1.0, /
&wt type='END' /
LISTOUT=POUT
DISANG=RST
and 2000 steps energy minimization with NMR constraints in
vacuum
&cntrl
ntwe = 5, ntwx = 5, ntpr = 5,
scnb = 2.0, scee = 1.2, nsnb = 25,
dielc = 1, cut = 15.0,
ntb = 0,
maxcyc =2000, ntmin = 1, ncyc = 500,
dx0 = 0.01, drms = 0.01,
ntr = 0,
imin = 1, nmropt = 1,
/
&wt type='REST',
istep1=0,istep2=2000,value1=1.0,
value2=1.0, /
&wt type='END' /
LISTOUT=POUT
DISANG=RST
REFINEMET IN EXPLICIT SOLVENT
For the explicit water refinement, the last structures were used
as initial structures. A solvent box whose edges are 10 A from the
closest proteins atoms was added to each structure. I am using TIP3P
water model and amber 99 FF for protein atoms. The protocol continues
as follows:
40 ps of MD with constant volume, gradually heating up from 0 K
to 50 K with 5 kcal/(mol A2 ) harmonic constraints on the
protein to their starting structures .
&cntrl
nstlim = 20000, dt = 0.002
ntwe = 500, ntwx = 500, ntpr = 50,
scnb = 2.0, scee = 1.2, nsnb = 25, dielc = 1, cut = 10.0,
ntb = 1,
ntr = 1,
ntc = 2, ntf = 2,
ntx = 1, irest = 0,
imin = 0, nmropt = 0,
ntt=1, tempi=0.0, temp0=50.0, tautp=1.0,
/
Keep protein fixed with weak restraints
5.0
RES 1 36
END
END
40 ps of MD with constant volume, gradually heating up from 50
K to 100 K with 5 kcal/(mol A2 ) harmonic constraints on
the protein to their starting structures.
&cntrl
nstlim = 20000, dt = 0.002
ntwe = 500, ntwx = 500, ntpr = 50,
scnb = 2.0, scee = 1.2, nsnb = 25,
dielc = 1, cut = 10.0,
ntb = 1,
ntr = 1,
ntc = 2, ntf = 2,
ntx = 1, irest = 0,
imin = 0, nmropt = 0,
ntt=1, tempi=50.0, temp0=100.0,
tautp=1.0,
/
Keep protein fixed with weak
restraints
5.0
RES 1 36
END
END
40 ps of MD with constant volume, gradually heating up from 100
K to 300 K with 5 kcal/(mol A2 ) harmonic constraints on
the protein to their starting structures
&cntrl
nstlim = 20000, dt = 0.002
ntwe = 500, ntwx = 500, ntpr = 50,
scnb = 2.0, scee = 1.2, nsnb = 25, dielc = 1, cut = 10.0,
ntb = 1,
ntr = 1,
ntc = 2, ntf = 2,
ntx = 1, irest = 0,
imin = 0, nmropt = 0,
ntt=1, tempi=100.0, temp0=300.0, tautp=1.0,
/
Keep protein fixed with weak restraints
5.0
RES 1 36
END
END
This was followed by 2 cycles of 40 ps constant pressure MD
with pressure relaxation time of 0.2 ps and 1.0 ps , with 5
kcal/(mol A2 ) harmonic constraints on the protein, at 300
K.
&cntrl
nstlim = 20000, dt = 0.002
ntwe = 500, ntwx = 500, ntpr = 50,
scnb = 2.0, scee = 1.2, nsnb = 25, dielc = 1, cut = 10.0,
ntb = 2,
ntr = 1,
ntc = 2, ntf = 2,
ntx = 1, irest = 0,
imin = 0, nmropt = 0,
ntt=1, tempi=300.0, temp0=300.0, tautp=1.0,
ntp=1, taup=0.2,
/
Keep protein fixed with weak restraints
5.0
RES 1 36
END
END
Then another 40 ps constant volume MD with 5 kcal/(mol A2
) harmonic constraints at 300K
&cntrl
nstlim = 20000, dt = 0.002
ntwe = 500, ntwx = 500, ntpr = 50,
scnb = 2.0, scee = 1.2, nsnb = 25, dielc = 1, cut = 10.0,
ntb = 1,
ntr = 1,
ntc = 2, ntf = 2,
ntx = 1, irest = 0,
imin = 0, nmropt = 0,
ntt=1, tempi=300.0, temp0=300.0, tautp=1.0,
/
Keep protein fixed with weak restraints
5.0
RES 1 36
END
END
and 60 ps constant volume MD with 0.5 kcal/(mol A2 )
&cntrl
nstlim = 30000, dt = 0.002
ntwe = 500, ntwx = 500, ntpr = 50,
scnb = 2.0, scee = 1.2, nsnb = 25, dielc = 1, cut = 10.0,
ntb = 1,
ntr = 1,
ntc = 2, ntf = 2,
ntx = 1, irest = 0,
imin = 0, nmropt = 0,
ntt=1, tempi=300.0, temp0=300.0, tautp=1.0,
/
Keep protein fixed with weak restraints
0.5
RES 1 36
END
END
Finally, a 100 ps MD simulation without harmonic constraints
was carried out at 300 K and NMR constraints.
&cntrl
nstlim = 100000, dt = 0.001
ntwe = 500, ntwx = 500, ntpr = 50,
scnb = 2.0, scee = 1.2, nsnb = 25, dielc = 1, cut = 10.0,
ntb = 1,
ntr = 0,
ntc = 2, ntf = 2,
ntx = 1, irest = 0,
imin = 0, nmropt = 1,
ntt=1, tempi=300.0, temp0=300.0, tautp=1.0,
/
&wt type='REST', istep1=0,istep2=100000,value1=1.0,
value2=1.0, /
&wt type='END' /
LISTOUT=POUT
DISANG=RST
After the final MD cycle, water molecules were removed from the
PDB file, and the protein molecule were subjected to a 2000 step
energy minimization in GB mode with NMR restraints.
&cntrl
igb = 2, saltcon=0.2, gbsa=1, cut=300.0,
intdiel=1.0, extdiel=78.5,
maxcyc = 2000, ntmin = 1, ncyc = 100, dx0 = 0.01, drms = 0.0001,
ntr = 0, ntpr=10, ntx=1, ntb=0,
imin = 1, nmropt = 1,
/
&wt type='REST', istep1=0,istep2=2000,value1=1.0,
value2=1.0, /
&wt type='END' /
LISTOUT=POUT
DISANG=RST
The trouble was I expected improve the RMSD between energy minimized structures respect the CYANA structures but this was not the outcome, maybe the parameters that I entered were not good values.
On the other side I want
to know if there are another protocol that you suggest me to perform
NMR refinement in explicit solvent because the last one takes about
120 h of CPU time per structure in a Pentium V processor.
Thanks for your
attention and time
Rogelio A. Hernández-López
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Received on Wed Sep 10 2008 - 06:07:49 PDT
