Dear All,
I am trying to minimize a glycosylated metalloprotein and prepared the prmtop and rst7 using tleap and forcefield parameters of the active site zinc ion coordination from Bras et al (2014) dx.doi.org/10.1021/cs500093h; ACS Catal. 2014, 4, 2587−2597. The Zn is coordinated to a water molecule, 2 histidines and a glutamate.
tleap gave warnings about missing improper torsion terms and I noticed that the glutamate OE1 is amongst them (which I'm guessing is alright?). The histidine NE2's are not mentioned. Parmed.py checks out fine after SetMolecules was used and the output from printDetails of metal site residues also looks fine.
After centering the molecule with cpptraj these files were used as input for minimization.
Here is my frcmod file (NK is for HIR361 (Histidine), NN is for HIT365 (Histidine), ON is for GLR389 (glutamate) and OK for WAR (TIP3P water):
REMARK, FORCEFIELD PARAMETERS OF ZINC SITE TAKEN FROM BRAS ET AL
MASS
NK 14.01 sp2 N in 5 memb.ring w/LP (HIS,ADE,GUA)
NN 14.01 sp2 N in 5 memb.ring w/LP (HIS,ADE,GUA)
ON 16.00 carboxyl and phosphonate group oxygen
OK 16.00 oxygen in tip3p water
Z1 65.40 zinc ion
BOND
NK-CR 488.0 1.335 JCC,7,(1986),230; HIS
NK-Z1 106.49 1.997 Bras
CV-NK 410.0 1.394 JCC,7,(1986),230; HIS
NN-CR 488.0 1.335 JCC,7,(1986),230; HIS
NN-Z1 88.07 2.029 Bras
CV-NN 410.0 1.394 JCC,7,(1986),230; HIS
ON-Z1 105.73 1.959 Bras
ON-C 656.0 1.250 JCC,7,(1986),230;GLU,ASP
OK-Z1 66.54 2.056 Bras
OK-HW 553.0 0.9572 TIP3P water
ANGL
NK-Z1-NN 36.12 112.53 Bras
NK-Z1-OK 17.73 137.14 Bras
NK-Z1-ON 64.70 95.65 Bras
NN-Z1-ON 26.27 115.04 Bras
NN-Z1-OK 26.93 99.83 Bras
OK-Z1-ON 46.96 95.20 Bras
CV-NN-Z1 26.5 130.48 Tuccinardi,2006 Angle Bras
CR-NN-Z1 26.5 122.56 Tuccinardi,2006 Angle Bras
CR-NK-Z1 26.5 113.83 Tuccinardi,2006 Angle Bras
CV-NK-Z1 26.5 134.21 Tuccinardi,2006 Angle Bras
Z1-OK-HW 35.47 92.69 Bras
C -ON-Z1 27.59 117.23 Bras
H4-CV-NN 50.0 120.00 amber
NN-CV-CC 70.0 120.00 amber
NN-CR-H5 50.0 120.00 amber
NN-CR-NA 70.0 120.00 amber
CV-NN-CR 70.0 117.00 amber
H4-CV-NK 50.0 120.00 amber
NK-CV-CC 70.0 120.00 amber
NK-CR-H5 50.0 120.00 amber
NK-CR-NA 70.0 120.00 amber
CV-NK-CR 70.0 117.00 amber
CT-C -ON 70.0 117.00 amber
O2-C -ON 80.0 126.00 amber (AA GLU(SCH JPC 79,2379))
HW-OK-HW 100.0 104.52 amber TIP3p water
HW-HW-OK 0.0 127.74 amber (crystallographic water with 3 bonds)
DIHE
CC-CV-NN-Z1 1 0.0 180.0 1.0
NA-CR-NN-Z1 1 0.0 180.0 1.0
CR-NN-Z1-NK 1 0.0 180.0 1.0
CR-NN-Z1-OK 1 0.0 180.0 1.0
H5-CR-NN-Z1 1 0.0 180.0 1.0
NN-Z1-NK-CV 1 0.0 180.0 1.0
NN-Z1-NK-CR 1 0.0 180.0 1.0
NN-Z1-OK-CV 1 0.0 180.0 1.0
NN-Z1-OK-CR 1 0.0 180.0 1.0
CC-CV-NK-Z1 1 0.0 180.0 1.0
NA-CR-NK-Z1 1 0.0 180.0 1.0
CR-NK-Z1-ON 1 0.0 180.0 1.0
H5-CR-NK-Z1 1 0.0 180.0 1.0
CR-NK-Z1-OK 1 0.0 180.0 1.0
NK-Z1-NN-CV 1 0.0 180.0 1.0
NK-Z1-ON-C 1 0.0 180.0 1.0
NK-Z1-OK-HW 1 0.0 180.0 1.0
CV-NK-Z1-ON 1 0.0 180.0 1.0
CV-NK-Z1-OK 1 0.0 180.0 1.0
H4-CV-NK-Z1 1 0.0 180.0 1.0
CR-NN-Z1-ON 1 0.0 180.0 1.0
NN-Z1-ON-C 1 0.0 180.0 1.0
NN-Z1-OK-HW 1 0.0 180.0 1.0
CV-NN-Z1-ON 1 0.0 180.0 1.0
CV-NN-Z1-OK 1 0.0 180.0 1.0
H4-CV-NN-Z1 1 0.0 180.0 1.0
CT-C-ON-Z1 1 0.0 180.0 1.0
C-ON-Z1-OK 1 0.0 180.0 1.0
ON-Z1-OK-HW 1 0.0 180.0 1.0
O2-C-ON-Z1 1 0.0 180.0 1.0
Z1-OK-HW-HW 1 0.0 180.0 1.0
CR-NN-CV-CC 2 4.80 180.0 2.0
CV-NN-CR-H5 2 10.00 180.0 2.0
CV-NN-CR-NA 2 10.00 180.0 2.0
H4-CV-NN-CR 2 4.80 180.0 2.0
CR-NK-CV-CC 2 4.80 180.0 2.0
CV-NK-CR-H5 2 10.00 180.0 2.0
CV-NK-CR-NA 2 10.00 180.0 2.0
H4-CV-NK-CR 2 4.80 180.0 2.0
IMPR
CR-CV-Z1-NN 1 50.0 180.0 2.
CV-CR-Z1-NN 1 50.0 180.0 2.
CR-CV-Z1-NK 1 50.0 180.0 2.
CV-CR-Z1-NK 1 50.0 180.0 2.
CV-CR-NK-Z1 1 500.0 180.0 2.
CV-CR-NN-Z1 1 500.0 180.0 2.
NONB
NK 1.8240 0.1700 Amber
NN 1.8240 0.1700 Amber
ON 1.6612 0.2100 Amber
OK 1.7683 0.1520 Amber TIP3p water
Z1 1.7000 0.0125 Amber (Ryde et al)
I first minimized the metal site without SHAKE since my starting geometry did not exactly match the frcmod (min1). This worked out fine.
Constant Volume Minimization 1 with all but metal site fixed SD only
# Control section
&cntrl
imin=1,
irest = 0,
ntmin = 2, maxcyc = 500, dx0 = 0.01, drms = 0.0001,
cut = 10.0,
ntb = 1,
ntp = 0,
ntwx = 20,
ntpr = 20,
ntwr = 500,
ioutfm = 1,
iwrap = 1,
ntr = 1, restraintmask='!:WAR,ZN1,HIR,HIT,GLR', restraint_wt=50.0
/
Thereafter I tried minimizing the solvent with the solute atoms restrained.
Constant Volume Minimization 2 of solvent
# Control section
&cntrl
imin=1,
irest = 0,
ntmin = 1, maxcyc = 5000, ncyc = 2500, dx0 = 0.01, drms = 0.0001,
cut = 10.0,
ntb = 1,
ntp = 0,
ntwx = 50,
ntpr = 50,
ntwr = 5000,
ioutfm = 1,
iwrap = 1,
ntr = 1, restraintmask='!:WAT,Na+', restraint_wt=50.0
/
There are no errors but I noticed that despite the restraints the metal site's HIR and HIT NE2 atoms still move. They have bonds to the ZN and get pulled so that the histidine ring loses some planarity. Although I do see the waters moving slightly, for the most part in the min2.out the gmax is for these NE2 atoms and they are quite mobile. I'm worried that the minimizer is trying to optimize these atoms and not minimizing the solvent properly. Could someone please explain to me why they still move despite the restraint?
Although tleap did not detect missing improper torsion terms for the NK and NN atoms of these histidines I think that perhaps the ones given in the frcmod file are not being read properly. Improper torsion terms would maintain the planarity of the sp2 bonds in the histidine rings right?
I obtained this frcmod from the authors and noticed that the spacing of these lines differ. How could I confirm that they are being read? If this is not causing the strange behaviour what else could I check?
Kind regards
Lizelle Lubbe
PhD (Chemical Biology) candidate
Department of Integrative Biomedical Sciences
University of Cape Town
South Africa
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Received on Mon Sep 18 2017 - 09:30:02 PDT
