Plese find below the requested "in" scripts and the corresponding final
"out" results.
*min1.in <
http://min1.in>*
initial minimisation solvent + ions
&cntrl
imin = 1, iwrap = 1,
maxcyc = 2000,
ncyc = 1000,
ntb = 1,
ntr = 1,
cut = 10.0
/
Hold the protein fixed
50.0
RES 1 190
END
END
Maximum number of minimization cycles reached.
FINAL RESULTS
NSTEP ENERGY RMS GMAX NAME NUMBER
2000 -2.1093E+05 2.8232E-01 4.0307E+01 CD 1325
BOND = 15957.8646 ANGLE = 574.0886 DIHED = 2123.4007
VDWAALS = 38759.9432 EEL = -275672.7908 HBOND = 0.0000
1-4 VDW = 636.5620 1-4 EEL = 6467.6717 RESTRAINT = 221.6106
EAMBER = -211153.2601
*min2.in <
http://min2.in>*
initial minimisation whole system
&cntrl
imin = 1,
maxcyc = 4000,
ncyc = 2000,
ntb = 1, iwrap = 1,
ntr = 0,
cut = 10.0
nmropt=1,
/
&wt type='END' /
DISANG= ../RST.dist
LISTOUT=POUT
/
Maximum number of minimization cycles reached.
FINAL RESULTS
NSTEP ENERGY RMS GMAX NAME NUMBER
4000 -2.1551E+05 6.2643E-02 2.6697E+00 C 1945
BOND = 16703.5751 ANGLE = 464.5674 DIHED = 2041.2629
VDWAALS = 41783.9681 EEL = -283555.5329 HBOND = 0.0000
1-4 VDW = 606.2511 1-4 EEL = 6446.1250 RESTRAINT = 0.9745
EAMBER = -215509.7832
NMR restraints: Bond = 0.974 Angle = 0.000 Torsion = 0.000
===============================================================================
------------------------------------------------------------------------------
Final Restraint Analysis for coords: mc_min2.rst
Restraints, deviations, and energy contributions: pencut = 0.10
------------------------------------------------------------------------------
First atom Last atom curr. value target deviation penalty
------------------------------------------------------------------------------
OG SER 66 -- C ALA 5: 3.137 3.000 0.137 0.941 d 0: 0
Total distance penalty: 0.974
------------------------------------------------------------------------------
*md1.in <
http://md1.in>*
heating phase
&cntrl
imin = 0,
irest = 0,
ntx = 1,
ntb = 1,
cut = 10.0,
ntr = 1,
ntc = 2,
ntf = 2,
tempi = 10.0,
temp0 = 310.0,
ntt = 3,
gamma_ln = 1.0,
nstlim = 50000, dt = 0.002, iwrap = 1,
ntpr = 100, ntwx = 100, ntwr = 1000, ig = -1,
/
Keep the solute fixed with weak restraints
10.0
RES 1 190
END
END
A V E R A G E S O V E R 500 S T E P S
NSTEP = 50000 TIME(PS) = 100.000 TEMP(K) = 306.86 PRESS = 0.0
Etot = -124514.2131 EKtot = 32469.6641 EPtot = -156983.8772
BOND = 583.9065 ANGLE = 1510.2495 DIHED = 2244.4085
1-4 NB = 672.3453 1-4 EEL = 6426.1792 VDWAALS = 19989.5543
EELEC = -188994.7508 EHBOND = 0.0000 RESTRAINT = 584.2303
EAMBER (non-restraint) = -157568.1075
------------------------------------------------------------------------------
R M S F L U C T U A T I O N S
NSTEP = 50000 TIME(PS) = 100.000 TEMP(K) = 19.51 PRESS = 0.0
Etot = 5441.1086 EKtot = 2064.0676 EPtot = 3396.1267
BOND = 34.8406 ANGLE = 70.4932 DIHED = 17.2165
1-4 NB = 8.8790 1-4 EEL = 22.5288 VDWAALS = 1233.2218
EELEC = 4476.3952 EHBOND = 0.0000 RESTRAINT = 39.7603
EAMBER (non-restraint) = 3356.3664
------------------------------------------------------------------------------
*md2.in <
http://md2.in>*
200ps MD
&cntrl
imin = 0, irest = 1, ntx = 5,
ntb = 2, pres0 = 1.0, ntp = 1, iwrap = 1,
taup = 2.0,
cut = 10.0, ntr = 0,
ntc = 2, ntf = 2,
temp0 = 310.0,
ntt = 3, gamma_ln = 1.0,
nstlim = 100000, dt = 0.002,
ntpr = 100, ntwx = 100, ntwr = 1000, ig = -1,
nmropt=1,
/
&wt type='END' /
DISANG= ../RST.dist
LISTOUT=POUT
/
A V E R A G E S O V E R 1000 S T E P S
NSTEP = 100000 TIME(PS) = 300.000 TEMP(K) = 309.97 PRESS = -17.8
Etot = -127511.1996 EKtot = 32799.2545 EPtot = -160310.4541
BOND = 612.0199 ANGLE = 1684.3452 DIHED = 2332.6916
1-4 NB = 703.7638 1-4 EEL = 6451.8025 VDWAALS = 21018.2055
EELEC = -193114.3535 EHBOND = 0.0000 RESTRAINT = 1.0708
EAMBER (non-restraint) = -160311.5249
EKCMT = 15274.2930 VIRIAL = 15489.4872 VOLUME = 534336.9303
Density = 0.9891
------------------------------------------------------------------------------
NMR restraints: Bond = 2.399 Angle = 0.000 Torsion = 0.000
===============================================================================
R M S F L U C T U A T I O N S
NSTEP = 100000 TIME(PS) = 300.000 TEMP(K) = 1.41 PRESS = 135.8
Etot = 496.1809 EKtot = 149.3112 EPtot = 444.9143
BOND = 21.6080 ANGLE = 32.1149 DIHED = 18.0821
1-4 NB = 11.4605 1-4 EEL = 32.5536 VDWAALS = 213.7837
EELEC = 583.9388 EHBOND = 0.0000 RESTRAINT = 0.9930
EAMBER (non-restraint) = 443.9214
EKCMT = 99.8970 VIRIAL = 1617.9579 VOLUME = 4729.7524
Density = 0.0083
------------------------------------------------------------------------------
NMR restraints on final step:
------------------------------------------------------------------------------
Final Restraint Analysis for coords: mc_md2.rst
Restraints, deviations, and energy contributions: pencut = 0.10
------------------------------------------------------------------------------
First atom Last atom curr. value target deviation penalty
------------------------------------------------------------------------------
OG SER 66 -- C ALA 5: 3.225 3.000 0.225 2.532 d 0: 0
Total distance penalty: 2.532
------------------------------------------------------------------------------
*md3.in <
http://md3.in>*
30 ns production phase MD
&cntrl
imin = 0, irest = 1, ntx = 5,
ntb = 2, pres0 = 1.0, ntp = 1,
taup = 1.0,
cut = 10.0, ntr = 0, iwrap = 1,
ntc = 2, ntf = 2,
tempi = 310.0, temp0 = 310.0,
ntt = 3, gamma_ln = 0.5,
nstlim = 15000000, dt = 0.002,
ntpr = 5000, ntwx = 5000, ntwr = 5000, ig = -1,
nmropt=1,
/
&wt type='END' /
DISANG= ../RST.dist
LISTOUT=POUT
/
A V E R A G E S O V E R 3000 S T E P S
NSTEP = 15000000 TIME(PS) = 30300.000 TEMP(K) = 310.00 PRESS = 1.1
Etot = -127695.4330 EKtot = 32801.8774 EPtot = -160497.3104
BOND = 609.8495 ANGLE = 1679.0266 DIHED = 2333.8034
1-4 NB = 702.1031 1-4 EEL = 6453.2305 VDWAALS = 21020.2076
EELEC = -193296.3975 EHBOND = 0.0000 RESTRAINT = 0.8663
EAMBER (non-restraint) = -160498.1768
EKCMT = 15278.3467 VIRIAL = 15265.5129 VOLUME = 532997.6037
Density = 0.9915
------------------------------------------------------------------------------
NMR restraints: Bond = 0.000 Angle = 0.000 Torsion = 0.000
===============================================================================
R M S F L U C T U A T I O N S
NSTEP = 15000000 TIME(PS) = 30300.000 TEMP(K) = 1.33 PRESS = 93.3
Etot = 249.4287 EKtot = 140.3800 EPtot = 201.0937
BOND = 21.4083 ANGLE = 31.8763 DIHED = 18.7948
1-4 NB = 11.3473 1-4 EEL = 36.1648 VDWAALS = 179.7637
EELEC = 299.9844 EHBOND = 0.0000 RESTRAINT = 0.8820
EAMBER (non-restraint) = 200.2117
EKCMT = 94.8748 VIRIAL = 1080.6558 VOLUME = 632.8248
Density = 0.0012
------------------------------------------------------------------------------
NMR restraints on final step:
------------------------------------------------------------------------------
Final Restraint Analysis for coords: mc_md3.rst
Restraints, deviations, and energy contributions: pencut = 0.10
------------------------------------------------------------------------------
First atom Last atom curr. value target deviation penalty
------------------------------------------------------------------------------
------------------------------------------------------------------------------
*****************************
Michael Shokhen, PhD
Associate Professor
Department of Chemistry
Bar Ilan University,
Ramat Gan, 52900
Israel
email: michael.shokhen.gmail.com
email: shokhen.mail.biu.ac.il
On Sun, Jul 26, 2015 at 9:24 PM, Carlos Simmerling <
carlos.simmerling.gmail.com> wrote:
> I haven't seen your minimization and equilibration inputs, just the
> production phase. the problem probably starts much earlier. you need to
> look at the minimization right after you add the peptide. what are the
> final energies, force and gmax? just because the minimization finishes does
> not mean that there are not bad contacts that it could not fix. those often
> show up as unwanted structural changes in the resulting MD. look carefully
> at the equilibration and see what things look like before you run
> production MD.
>
> On Sun, Jul 26, 2015 at 2:21 PM, Michael Shokhen <
> michael.shokhen.biu.ac.il>
> wrote:
>
> > Hi Carlos,
> >
> > You absolutely right - generation of initial enzyme-substrate structure
> > is non-trivial task and could be the main source of all following errors.
> > Fortunately, I know several principal structural conditions that must be
> > satisfied at the construction
> > by initial modeling of enzyme-substrate complex known from available
> > experiments,
> > aa sequence of peptide substrate influencing its recognition alignment
> in
> > the enzyme active site, 3D structure of free enzyme,
> > general structural features common for serine protease catalysis, etc.
> > Of course there are still many unknown things that could be identified by
> > try and error
> > simulations only.
> > You wrote: "a crucial step is the energy/forces at the start and end of
> the
> > minimization phase after adding the peptide."
> > Of course I used previous minimization and the following initial MD
> > structural relaxation
> > before starting the productive MD simulations. All energy conditions were
> > well satisfied
> > and the enzyme-substrate complex was stable energetically.
> > Sorry bother you again: Are my scripts correct or there are errors?
> >
> > Thank you,
> > Michael
> >
> >
> > *****************************
> > Michael Shokhen, PhD
> > Associate Professor
> > Department of Chemistry
> > Bar Ilan University,
> > Ramat Gan, 52900
> > Israel
> > email: shokhen.mail.biu.ac.il
> >
> > ________________________________________
> > From: Carlos Simmerling <carlos.simmerling.gmail.com>
> > Sent: Sunday, July 26, 2015 8:49 PM
> > To: AMBER Mailing List
> > Subject: Re: [AMBER] MD simulation: cis isomer of peptide bond
> >
> > Hi Michael,
> > how did you add the structure of the peptide substrate? also as I
> mentioned
> > before, a crucial step is the energy/forces at the start and end of the
> > minimization phase after adding the peptide. if for some reason the
> > structure still has high forces, or the restraints cannot be satisfied,
> > then you will have subsequent problems that many manifest as the
> > isomerization. if you restrain that, it may simply do something else
> > non-physical. I would focus on figuring out why it happens, not just
> trying
> > to keep it from happening. unless your structure is highly strained I
> would
> > not expect this behavior (except if there is physical justification for
> > stability of the cis isomer...)
> > carlos
> >
> > On Sun, Jul 26, 2015 at 1:34 PM, Michael Shokhen <
> > michael.shokhen.biu.ac.il>
> > wrote:
> >
> > > Hi Carlos,
> > >
> > > Many thanks for your reply.
> > > The initial 3D structure of enzyme was
> > > generated by homology modeling based on
> > > the known aa sequence. Then the structure of free enzyme
> > > was relaxed by amber ff14sb in water box at about 300 ns productive
> > > MD simulation until RMSD convergence of the backbone was achieved.
> > > It is serine protease where catalytic Ser nucleophile is activated by
> > > His general base catalysis. In the Michaelis complex of serine protease
> > > N eps His and O gamma Ser form H-bond (the ultimate condition of
> > > proton transfer), so I used 2.8 A restraint of this interatomic
> distance.
> > > The second restraint is between attacking Ser O gamma atom and peptide
> > > substrate
> > > amide carbonyl carbon atom (electrophilic center). This distance was
> > > restraint
> > > at 3.0 A in the MD simulated Michaelis complex (MC). The mentioned
> > > distances restraint
> > > is what I called as "biochemically reasonable". I used the restraints
> > > because without them the ff14sb didn't keep the mentioned critical for
> > > catalysis
> > > distances at reasonable values considerably increasing them.
> > > I have been planning to relax by unrestrained MD the MC structure when
> > > previous conditional
> > > restraint MD simulation would satisfy RMSD. Unfortunately, as I wrote I
> > > faced cis
> > > isomerization of peptide bond in restrained MD.
> > >
> > > I still need your expertize of my script files attached to previous
> > email.
> > >
> > > Thank you,
> > > Michael
> > >
> > >
> > >
> > > *****************************
> > > Michael Shokhen, PhD
> > > Associate Professor
> > > Department of Chemistry
> > > Bar Ilan University,
> > > Ramat Gan, 52900
> > > Israel
> > > email: shokhen.mail.biu.ac.il
> > >
> > > ________________________________________
> > > From: Carlos Simmerling <carlos.simmerling.gmail.com>
> > > Sent: Sunday, July 26, 2015 7:43 PM
> > > To: AMBER Mailing List
> > > Subject: Re: [AMBER] MD simulation: cis isomer of peptide bond
> > >
> > > the barrier for this is fairly large so I would not expect it unless
> you
> > > have some other problem in your structure, or the cis isomer is
> strongly
> > > stabilized during/after the transition. I would look to see what's
> going
> > on
> > > right before it happens. using a restraint may just cover up the larger
> > > problem. it might be in the initial structure (you didn't say how you
> > > obtained it) or perhaps in the distance restraints that you applied.
> what
> > > do you mean by "biochemically reasonable"? look at the energies and
> force
> > > in your minimizations prior to MD.
> > >
> > > On Sun, Jul 26, 2015 at 12:07 PM, Michael Shokhen <
> > > michael.shokhen.biu.ac.il
> > > > wrote:
> > >
> > > > Dear Amber List members,
> > > >
> > > > I have been running production MD simulation of non-covalent enzyme
> > > > –peptide
> > > > substrate (SVLAKEL) complex by Amber14 with ff14sb force field in
> > > periodic
> > > > water box.
> > > > I used restraints on two interatomic distances (RST.dist file) in
> order
> > > to
> > > > keep
> > > > biochemically reasonable position of catalytic residues and
> substrate.
> > > > Suddenly,
> > > > at 31 ns snapshot I have observed irrelevant isomerization at the A-K
> > > > (Ala-Lys)
> > > > amide bond of peptide substrate backbone where carbonyl bond of Ala
> > > became
> > > > in cis position to N-H amide bond of Lys.
> > > >
> > > > In order to fix the problem I have added torsional restraint on the
> > > > problematic
> > > > amide bond (rst.bb file) and restarted MD from 30 ns snapshot with
> > > correct
> > > > trans isomer of the target amide bond. Unfortunately, I found again
> > > > incorrect cis
> > > > isomer at 31 ns on this amide bond.
> > > > It seems that there is an error in my md3.in and rst.bb files.
> > > > See below listings of the files.
> > > >
> > > > I would appreciate your help.
> > > >
> > > > Thank you,
> > > >
> > > > Michael
> > > >
> > > > *md4.in <http://md4.in>*
> > > >
> > > > 30 ns production phase MD
> > > > &cntrl
> > > > imin = 0, irest = 1, ntx = 5,
> > > > ntb = 2, pres0 = 1.0, ntp = 1,
> > > > taup = 1.0,
> > > > cut = 10.0, ntr = 0, iwrap = 1,
> > > > ntc = 2, ntf = 2,
> > > > tempi = 310.0, temp0 = 310.0,
> > > > ntt = 3, gamma_ln = 0.5,
> > > > nstlim = 15000000, dt = 0.002,
> > > > ntpr = 5000, ntwx = 5000, ntwr = 5000, ig = -1,
> > > > nmropt=1,
> > > > /
> > > > &wt type='END' /
> > > > DISANG= ../RST.dist
> > > > LISTOUT=POUT
> > > > /
> > > > DISANG1= ../rst.bb
> > > > LISTOUT=POUT
> > > > /
> > > >
> > > >
> > > > *RST.dist*
> > > >
> > > > &rst
> > > > ixpk= 0, nxpk= 0, iat=1030,1692, r1= 1.30, r2= 1.80, r3= 2.80, r4=
> > > 3.30,
> > > > rk2=50.0, rk3=50.0, ir6=1, ialtd=0,
> > > > &end
> > > > &rst
> > > > ixpk= 0, nxpk= 0, iat=1030,61, r1= 1.30, r2= 1.80, r3= 3.00, r4=
> > 3.50,
> > > > rk2=50.0, rk3=50.0, ir6=1, ialtd=0,
> > > > &end
> > > >
> > > >
> > > > *rst.bb <http://rst.bb>*
> > > >
> > > > &rst
> > > > iat=36,51,53,55, r1=0., r2=180., r3=180., r4=360.,
> > > > rk2 = 35., rk3 = 35., /
> > > >
> > > >
> > > >
> > > >
> > > > *****************************
> > > > Michael Shokhen, PhD
> > > > Associate Professor
> > > > Department of Chemistry
> > > > Bar Ilan University,
> > > > Ramat Gan, 52900
> > > > Israel
> > > > email: michael.shokhen.gmail.com
> > > > email: shokhen.mail.biu.ac.il
> > > > _______________________________________________
> > > > AMBER mailing list
> > > > AMBER.ambermd.org
> > > > http://lists.ambermd.org/mailman/listinfo/amber
> > > >
> > > _______________________________________________
> > > AMBER mailing list
> > > AMBER.ambermd.org
> > > http://lists.ambermd.org/mailman/listinfo/amber
> > >
> > > _______________________________________________
> > > AMBER mailing list
> > > AMBER.ambermd.org
> > > http://lists.ambermd.org/mailman/listinfo/amber
> > >
> > _______________________________________________
> > AMBER mailing list
> > AMBER.ambermd.org
> > http://lists.ambermd.org/mailman/listinfo/amber
> >
> > _______________________________________________
> > AMBER mailing list
> > AMBER.ambermd.org
> > http://lists.ambermd.org/mailman/listinfo/amber
> >
> _______________________________________________
> AMBER mailing list
> AMBER.ambermd.org
> http://lists.ambermd.org/mailman/listinfo/amber
>
_______________________________________________
AMBER mailing list
AMBER.ambermd.org
http://lists.ambermd.org/mailman/listinfo/amber
Received on Sun Jul 26 2015 - 12:30:02 PDT
