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
> >
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Received on Sun Jul 26 2015 - 11:30:02 PDT
