Section 1: Preparing the PDB file

1EMA是本次教程所用的pdb,可以在PDB数据库下载。

pdb4amber -i 1EMA.pdb -o gfp.pdb --dry --reduce

pdb4amber命令用于amber输入pdb格式文件的准备。 --dry会删除晶体结构中的水分子(WATER),--reduce会对pdb加氢(H)。命令执行完成后需要对产生的gfp.pdb进行手工微调,1) 需要把文件中所有MSE更换为MET;2) 把所有SE 原子(Se)换为硫(SD)原子; 3)在pdb文件最后一列中,把SE元素,换乘S元素。

pdb4amber analyses PDB files and cleans them for further usage, especially with the LeaP programs of Amber. It does NOT use any information in the original PDB file other than that contained in ATOM and HETATM records. The final output files are stripped of everything not directly related to ATOM or HETATM records.

Section 2: Computing partial charges and atom types for CRO

gfp.pdb文件中的CRO残基为非标准氨基酸残基,不在标准amber残基库中,所以需要对此非标准氨基酸进行加电(deriving charges)和决定原子类型(determining the atom types of the CRO residue)操作;

对于非标准残基CRO,我们使用PDB数据库中的CIF文件对其进行定义,CRO.cif;

我们使用antechamber程序读取CRO.cif (ccif)并对其进行加电(-c bcc)及分配原子类型 (-at amber)操作,(For more general organic molecules, it is usually better to use gaff atom types.)

运行下面程序前,我们需要先将CRO.cif文件中第五行中的LINKING字符删掉,不然会报错。

antechamber -fi ccif -i CRO.cif -bk CRO -fo ac -o cro.ac -c bcc -at amber

antechamber: This is the most important program in the package. It can perform many file conversions, and can also assign atomic charges and atom types. As required by the input, antechamber executes the following programs: sqm (or,alternatively, mopac or divcon), atomtype, am1bcc, bondtype, espgen, respgen and prepgen. It typically produces many intermediate files; these may be recognized by their names, in which all letters are upper-case.

Running Errors:

1. "Residue CRO has a type of LINKING.  Quitting" 执行上述命令时,报错LINKING:

解决方法有两种:1) 删除文件CRO.cif中的LINKING字符。

        2) 使用“//”注释掉$AMBERHOME/AmberTools/src/antechamber 下的mmcif.c文件的第68行,"// exit(1);",然后make install,重新安装antechamber,即可解决问题。

2. 使用diff命令对比程序运行结果会和对照结果文件不同,涉及到第一个原子NT和N,手动把NT改为N。

Section 3: Preparing the residue library and force field parameters for use with LEaP

PDB数据库中下载的CRO.cif在经过antechamber处理后,变成一个拥有完整的H完整的电荷的分子,但是我们的gpf.pdb文件中的的CRO是以共价键方式与周围的氨基酸连接的,所以为了使经过加氢加电的CRO分子连接(CRO分子的NC端连接到pdb中)到gpf.pdb文件中,我们需要对CRO分子进行处理,把CRO分子的NC端的H去掉,并和pdb分子中相应的位置连接,我们使用prepgen程序处理:

prepgen -i cro.ac -o cro.prepin -m cro.mc -rn CRO

 Prepgen: Prepgen generates the prep input file from an ac file. By default, the program generates a mainchain itself. However, you may also specify the main-chain atoms in the main chain file. From this file, you can also specify which atoms will be deleted, and whether to do charge correction or not. In order to generate the amino-acid-like residue (this kind of residue has one head atom and one tail atom to be connected to other residues), you need a main chain file. Sample main chain files are in $AMBERHOME/dat/antechamber.

使用prepgen程序及包含NC端原子,主链侧链信息的主链(mc)文件,对cro.ac进行处理,得到处理后的cro.prepin文件,至此,我们便得到了非标准残基CRO的残基库及电荷信息;

接下来,我们使用parmchk2程序检查CRO分子(cro.prepin)的共价键(bonds, angles, and dihedrals)参数化情况,

parmchk2 -i cro.prepin -f prepi -o frcmod.cro -a Y \
-p $AMBERHOME/dat/leap/parm/parm10.dat

parmchk2: The parmchk2 program figures out what parameters will be needed and checks to see if they are in the standard files. If not, it tries to make educated guesses, and puts these new parameters into a file we are calling "frcmod.cro" here. -p flag specifies the parm10.dat file because it is the main parameter database for the force field we plan to use, ff14SB.

frcmod.cro文件中,有的行会标记“ATTN, need revision“,这种标记意味着,parmchk2程序不能在parm10数据库中找到相似的参数,我们把这些标记的行在frcmod.cro文件中删除,然后再在gaff.dat数据库中去寻找合适的参数。

grep -v "ATTN" frcmod.cro > frcmod1.cro # Strip out ATTN lines
parmchk2 -i cro.prepin -f prepi -o frcmod2.cro

至此,我们得到两个frcmod文件,即frcmod1.cro(parm10.dat,删除了ATTN)和frcmod2.cro(gaff.dat),用于下面的步骤。

Section 4: Creating the topology and coordinate files for simulation

我们使用之前步骤中的文件,准备拓扑文件及坐标文件。我们使用ff14SB力场,使用隐式溶剂模型(igb=8,并设置PBRadii默认值为mbondi3);首先我们加载cro.prepin文件,然后对于参数文件,我们先load frcmod2.cro再load frcmod1.cro,以确保所有gaff参数被parm10参数替换,然后再导入gfp.pdb结构文件,输出gfp.parm7和gfp.rst7文件。

$$$ tleap.in

source leaprc.protein.ff14SB
set default PBRadii mbondi3
loadAmberPrep cro.prepin
loadAmberParams frcmod2.cro
loadAmberParams frcmod1.cro
x = loadPDB gfp.pdb
saveAmberParm x gfp.parm7 gfp.rst7
quit
tleap -f tleap.in

Section 5: Simulations; minimization, heating, equilibration, and production

Minimization

以初始坐标为起点,对整个体系优化,排除一些不利的构像

$$$ min.in

 simple generalized Born minimization script
&cntrl
imin=, ntb=, maxcyc=, ntpr=, cut=., igb=,
/
sander -O -i min.in -p gfp.parm7 -c gfp.rst7 -o min1.out -r min1.rst7

Heating

以minimization的rst文件为起点,对整个体系加热,200ps加热时间,从10K到300K

$$$ heat.in

Implicit solvent initial heating mdin
&cntrl
imin=, irest=, ntx=,
ntpr=, ntwx=, nstlim=,
dt=0.002, ntt=, tempi=,
temp0=, gamma_ln=1.0, ig=-,
ntp=, ntc=, ntf=, cut=,
ntb=, igb=, ioutfm=, nmropt=,
/
&wt
TYPE='TEMP0', ISTEP1=, ISTEP2=,
VALUE1=10.0, VALUE2=300.0,
/
&wt TYPE='END' /
sander -O -i heat.in -p gfp.parm7 -c min1.rst7 -o heat.mdout \
-x heat.nc -r heat.rst7

Production

以上一步坐标为起点,进行MD run 100ns

$$$ md.in

Implicit solvent molecular dynamics
&cntrl
imin=, irest=, ntx=,
ntpr=, ntwx=, nstlim=,
dt=0.002, ntt=, tempi=,
temp0=, gamma_ln=1.0, ig=-,
ntp=, ntc=, ntf=, cut=,
ntb=, igb=, ioutfm=,
/
sander -O -i md.in -p gfp.parm7 -c heat.rst7 -o md1.mdout \
-x md1.nc -r md1.rst7
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