!"#$%&'( *$% !%+,-&./01.2'3&4&.1 5$3,(16 3$(15"('% 4+.

'3&5- -#"4+7

uiomacs veision 4.S.S































}ohn E. Keiiigan, Ph.B.
Associate Biiectoi, Bioinfoimatics
The Cancei Institute of New }eisey
The Robeit Woou }ohnson Neuical School
12u Albany Stieet
New Biunswick, N} u89uS

(7S2) 2SS-447S
keiiigjeumunj.euu
2
In this tutoiial we will stuuy the uynamics of the complex of benzamiuine with tiypsin, a
seiine piotease ielateu to the cascaue of pioteases iesponsible foi iegulation of bloou
coagulation. We will use the iecently publisheu x-iay stiuctuie (SATL.pub).|1j This
tutoiial iequiies uiomacs (http:www.giomacs.oig) suite of moleculai simulation
piogiams,|2j the Ambei Antechambei piogiam (AmbeiTools 1.S fiom
http:ambeimu.oig),|S, 4j 0CSF Chimeia (http:plato.cgl.ucsf.euuchimeia), vNB
(http:www.ks.uiuc.euuReseaichvmu), anu ACPYPE
(http:coue.google.compacpype).|Sj



Staiting fiom SATL.pub, we neeu to extiact out the piotein fiom the liganu. 0se the
following commanus in the unix shell.

8%1, 'AT0N ' SATL.pub > piotein.pub
8%1, 'BEN A' SATL.pub > liganu.pub

Isolate the Ca
2+
ion

8%1, 'BETATN 16S1 CA' SATL.pub > ca-ion.pub

Combine piotein anu CA ion using the unix "cat" commanu.

5'# piotein.pub ca-ion.pub > piotein2.pub

Piocess piotein2.pub with pub2gmx as follows .

,49:836 ;** ambei99sb ;* piotein2.pub ;$ tip.pub ;, tip.top ;<'#1% tipSp ;&8.=

Note we aie using the Ambei99SB foice fielu|6j anu the TIPSP watei mouel.|7j We will
piocess the liganu using 0CSF Chimeia to insuie we builu an amiuinium ion as
benzamiuine is chaigeu at physiological pB.

0pen liganu.pub in !"#$ "&'()*+.
uo to Tools > Stiuctuie Euiting > AuuB
Follow with Tools > Stiuctuie Euiting > Auu Chaige (Select AN1-BCC in uialogue box).

When finisheu the molecule shoulu anu oveiall foimal chaige of +1.uu. Note we aie using
the geneial ambei foicefielu (gaff)|4j anu AN1-BCC chaiges|8j foi the small molecule.
H
2
N NH
2
Benzamidinium ion
S
Save the file in mol2 foimat with filename ben.mol2. Next we will iun acpype using the
paitial atomic chaiges fiom the mol2 file.

Run ACPYPE .

'5,+,1 ;& ben.mol2 ;5 usei

All output is uumpeu into a uiiectoiy calleu "ben.acpype". Always check to make suie the
atom types assigneu make sense accoiuing to the ,-$$ atom type uesciiptions posteu at
http:ambeimu.oigantechambeigaff.html#atomtype anu copy of same in the appenuix
of this uocument.

Combine piotein with liganu cooiuinates .
8%1, ;= AT0N tip.pub ben.acpypeben_NEW.pub > complex.pub

5, ben.acpypeben_uNX.itp ben.itp

0sing any text euitoi (e.g. vi) auu #incluue "ben.itp" iight aftei the foicefielu in tip.top anu
auu "BEN 1" to the | molecules j section at the enu of tip.top.

; Incluue foicefielu paiameteis
#incluue "ambei99sb.fffoicefielu.itp"
#incluue "ben.itp"

.

| molecules j
; Compounu #mols
Piotein_chain_A 1
BEN 1

14&#5$.* ;9# octaheuion ;* complex.pub ;$ tip-b4solv.pub ;4 1.u
81.9$6 ;5, tip-b4solv.pub ;5- spc216.gio ;$ tip-b4ion.pub ;, tip.top

em.mup

define = -DFLEXIBLE
integrator = cg ; steep
nsteps = 200
constraints = none
emtol = 1000.0
nstcgsteep = 10 ; do a steep every 10 steps of cg
emstep = 0.01 ; used with steep
nstcomm = 1
coulombtype = PME
ns_type = grid
rlist = 1.0
rcoulomb = 1.0
4
rvdw = 1.4
Tcoupl = no
Pcoupl = no
gen_vel = no
nstxout = 0 ; write coords every # step
optimize_fft = yes

We will auu NaCl to concentiation of u.1S N in the peiiouic box anu make an oveiall
neutial system in teims of foimal chaige.

8%$3,, ;* em.mup ;5 tip-b4ion.pub ;, tip.top ;$ ion.tpi

81.&$. ;- ion.tpi ;$ tip-b4em.pub ;.1"#%'( ;5$.5 u.1S ;, tip.top ;8 ion.log

Select uioup 19 (S0L)

8%$3,, ;* em.mup ;5 tip-b4em.pub ;, tip.top ;$ em.tpi

34%". ;> ;41**.3 em

0ui iun finisheu with the following iesult .

Polak-Ribiere Conjugate Gradients converged to Fmax < 1000 in 63 steps
Potential Energy = -4.4418656e+05
Maximum force = 9.5519733e+02 on atom 2472
Norm of force = 4.4277103e+01

./0'1'/2 *)01*+'2)3 *42 - Beie we will peifoim 1uu ps of NvT (constant volume anu
tempeiatuie) ensemble uynamics to ielax the watei in the system while applying iestiaints
to the piotein.

pi.mup

define = -DPOSRES
integrator = md
nsteps = 50000
dt = 0.002
constraints = all-bonds
nstcomm = 10
ns_type = grid
rlist = 1.2
rcoulomb = 1.0
rcoulomb-switch = 0.9
rvdw = 1.0
vdwtype = shift
rvdw-switch = 0.9
coulombtype = PME-Switch
Tcoupl = v-rescale
tau_t = 0.1 0.1
tc-grps = protein non-protein
ref_t = 300 300
S
Pcoupl = no
Pcoupltype = isotropic
tau_p = 0.5
compressibility = 4.5e-5
ref_p = 1.0
gen_vel = yes
gen_temp = 300
gen_seed = -1
nstxout = 250 ; write coords every # step
nstenergy = 100
energygrps = Protein BEN SOL ;energy group monitoring
lincs-iter = 2
DispCorr = EnerPres
optimize_fft = yes

8%$3,, ;* pi.mup ;5 em.gio ;, tip.top ;$ pi.tpi

.$=", 34%". ;41**.3 pi ?

.*/3451'/2 *42 - Run at constant piessuie anu tempeiatuie (NPT).

mu.mup

integrator = md
nsteps = 250000
dt = 0.002
constraints = all-bonds
nstcomm = 10
ns_type = grid
rlist = 1.2
rcoulomb = 1.0
rcoulomb-switch = 0.9
rvdw = 1.0
vdwtype = shift
rvdw-switch = 0.9
coulombtype = PME-Switch
Tcoupl = v-rescale
tau_t = 0.1 0.1
tc-grps = protein non-protein
ref_t = 300 300
Pcoupl = parrinello-rahman
Pcoupltype = isotropic
tau_p = 0.5
compressibility = 4.5e-5
ref_p = 1.0
gen_vel = yes
gen_temp = 300
gen_seed = -1
nstxout = 500 ; write coords every # step
nstenergy = 100
energygrps = Protein BEN SOL ;energy group monitoring
lincs-iter = 2
DispCorr = EnerPres
optimize_fft = yes
6

8%$3,, ;* mu.mup ;5 pi.gio ;, tip.top ;$ mu.tpi

.$=", 34%". ;41**.3 mu ?

0ui Suu ps iun completeu in 1 houi anu 24 minutes using all 8 coies of a uual quau coie
Intel Xeon 2.8 uBz cpu woikstation.

@.'(+-&-

6207)51 1&) 1*+8)51/*9 :'04+;;9. Fiist conveit the tiajectoiy to compiesseu foimat anu
iemove pbc aitifacts.

#%A5$.> ;- mu.tpi ;* mu.tii ;$ mu.xtc ;,95 nojump

Select u (System). 0se a moleculai viewing piogiam like vNB
(http:www.ks.uiuc.euuReseaichvmu) to view the tiajectoiy (mu.xtc). 0sing vNB
loau pi.gio anu loau mu.xtc into pi.gio. Bisplay iesiuue IB S8, 189 anu 19S (iesiu S8 189
19S) in auuition to benzamiuine (iesname BEN).

Boes benzamiuine iemain in the active site pocket uuiing the couise of the Suu ps
simulation oi uoes it begin to slip out.

What iesiuue(s) uoes benzamiuine inteiact with uuiing the couise of the Suu ps
simulation. (<'21: In vNB use "piotein within 8 of iesname BEN" to view piotein atoms
within 8 of benzamiuine.)

"&)5= 7*/1)'2 >+5=>/2) ?@#A *)B)*)25) 1/ 1&) 5*901+; 01*4514*) C

8B%3- ;- em.tpi ;* mu.tii ;$ mu-bkbone.xvg

Select 4 (Backbone) both times.

7


D:+;4+1) 1&) 24(>)* /B &93*/E)2 >/230 >)1F))2 >)2G+('3'2) +23 1&) 7*/1)'2 /:)* 1&)
5/4*0) /B 1&) 1*+8)51/*9H

8B=9$.4 ;- mu.tpi ;* mu.xtc ;."3 /=93

Select BEN anu Piotein

6,3:,- ;* hbmap.xpm ;$ hbplot.eps ;%'&.9$< ieu


8
We hau about 4 hyuiogen bonus in existence on aveiage between BEN anu the Piotein
uuiing the couise of the simulation. See the uiomacs manual foi moie 8B=9$.4 tool
options.

D2)*E9 -2+;90'0. 0btain the shoit iange Lennaiu-}ones anu shoit iange Coulomb eneigies
of the BEN-Piotein inteiaction using the uiomacs 8B1.1%8+ eneigy analysis tool.

8B1.1%8+ ;- mu.tpi ;* mu.eui ;$ ben-tip-L}SR-CoulSR.xvg

Statistics over 250001 steps [ 0.0000 through 500.0000 ps ], 2 data sets
All statistics are over 25001 points

Energy Average Err.Est. RMSD Tot-Drift
-------------------------------------------------------------------------------
Coul-SR:Protein-BEN -190.533 0.88 15.0135 -3.43307 (kJ/mol)
LJ-SR:Protein-BEN -68.6689 1.3 11.0709 -6.82649 (kJ/mol)

Dint = <DL}> + <DCoul> = -68.7 + -19u.S = -2S9.2 k}mol = -62.u kcalmol

The Dint is a ciuue inteiaction eneigy baseu on shoit-iange eneigy components. This is not
a binuing eneigy. This numbei is a ciuue qualitative estimate of the stability of the Tiypsin-
benzamiuine complex.

You may also obtain othei infoimation fiom the eneigy file (.eui) like Piessuie,
Tempeiatuie, Kinetic Eneigy, etc.

C1*1%1.51-D

1. Yamane, }., et al., 62I5*901+; +BB'2'19 *+2='2E /B B*+E()21 &'1 5/(7/4230 *):)+;0 +
*);+1'/20&'7 F'1& 1&)'* '2&'>'1/*9 +51':'1')0H }. Appl. Ciystallogi., 2u11. EE: p. 798.
2. Bess, B., et al., ,?J@-"# KL -;E/*'1&(0 B/* &'E&;9 )BB'5')21M ;/+3I>+;+25)3M +23 05+;+>;)
(/;)54;+* 0'(4;+1'/2H }. Chem. Theoi. Comp., 2uu8. E(S): p. 4SS-447.
S. Wang, }., et al., -21)5&+(>)*M +2 +55)00/*9 0/B1F+*) 7+5=+E) B/* (/;)54;+* ()5&+2'50
5+;54;+1'/20H }. Nol. uiaphics, 2uu6. :F: p. 247-26u.
4. Wang, }., et al., A):);/7()21 +23 1)01'2E /B + E)2)*+; +(>)* B/*5) B');3H }. Comput.
Chem., 2uu4. :F(9): p. 11S7-1174.
S. Sousa Ba Silva, A.W., W.F. vianken, anu E.B. Laue, -21)"&+(>)* .N1&/2 .+*0)*
'21)*B+5DH, 2u1u.
6. Boinak, v., et al., "/(7+*'0/2 /B (4;1'7;) -(>)* B/*5) B');30 +23 3):);/7()21 /B
'(7*/:)3 7*/1)'2 >+5=>/2) 7+*+()1)*0H Pioteins, 2uu6. GF(S): p. 712-2S.
7. }oigensen, W., et al., "/(7+*'0/2 /B #'(7;) ./1)21'+; $4251'/20 B/* #'(4;+1'2E O'P4'3
Q+1)*H }. Chem. Phys., 198S. HI: p. 926-9SS.
8. }akalian, A., B.B. }ack, anu C.I. Bayly, $+01M )BB'5')21 E)2)*+1'/2 /B &'E&IP4+;'19 +1/('5
5&+*E)0H -@RIS"" (/3);L 66H .+*+()1)*'G+1'/2 +23 :+;'3+1'/2H }. Comput. Chem., 2uu2.
:J(16): p. 162S-41.

9

@,,1.4&6

The following table was obtained from http://ambermd.org/antechamber/gaff.html#atomtype . Table I lists the basic
(a) and special (b) atom types in GAFF.
Table I (a). Basic Atom Types in GAFF
Atom type Description
Atom
type
Description
c
c1
c2
c3
ca
sp2 C in C=O, C=S
sp1 C
sp2 C, aliphatic
sp3 C
sp2 C, aromatic
o
oh
os


sp2 O in C=O, COO-
sp3 O in hydroxyl group
sp3 O in ether and ester


n
n1
n2
n3
n4
na
nh
no
sp2 N in amide
sp1 N
sp2 N with 2 subst.
readl double bond
sp3 N with 3 subst.
sp3 N with 4 subst.
sp2 N with 3 subst
amine N connected
to the aromatic rings
N in nitro group
s2
sh
ss
s4
s6
sp2 S (p=S, C=S etc)
sp3 S in thiol group
sp3 S in -SR and SS
hypervalent S, 3 subst.
hypervalent S, 4 subst.
hc
ha
hn
ho
hs
hp
H on aliphatic C
H on aromatic C
H on N
H on O
H on S
H on P
f
cl
br
i
any F
any Cl
any Br
any I
p2
p3
p4
p5
sp2 P (C=P etc)
sp3 P, 3 subst.
hypervalent P, 3 subst.
hypervalent P, 4 subst.


Table I (b). Special Atom Types in GAFF
Atom type Description Atom type Description
h1
h2
h3
h4
h5


H on aliphatic C with 1 EW group;
H on aliphatic C with 2 EW group;
H on aliphatic C with 3 EW group;
H on aromatic C with 4 EW group;
H on aromatic C with 5 EW group;


cc(cd)
ce(cf)
cp(cq)
cu
cv
cx
cy
inner sp2 C in conj. ring systems
inner sp2 C in conj. chain systems
bridge aromatic C
sp2 C in three-memberred rings
sp2 C in four-memberred rings
sp3 C in three-memberred rings
sp3 C in four-memberred rings
n
nb
nc(nd)
sx
sy
aromatic nitrogen
inner sp2 N in conj. ring systems
inner sp2 N in conj. chain systems
conj. S, 3 subst.
conj. S, 4 subst.
pb
pc(pd)
pe(pf)
px
py
aromatic phosphorus
inner sp2 P in conj. ring systems
inner sp2 P in conj. chain systems
conj. P, 3 subst.
conj. P, 4 subst.
EW: electron-withdraw group

4. Wang, J., R. Wolf, J. Caldwell, P. Kollman, and D.A. Case, Development and testing of
a general amber force field. J. Comput. Chem., 2004. 25(9): p. 1157-1174.