Gromacs用alpha-A链和beta-B链制作电荷平衡的测试蛋白
如果直接将蛋白质不经过任何处理就作为输入使用insert插入，在进行到最后nvt平衡的grompp这一步就会报错初始能量过高，所以这里尝试使用离子平衡和能量最小化的方法先处理蛋白，然后再插入整体结构。
这里用chain-A和chain-B测试，具体结构是先将chain-A和chain-B溶于水，然后做离子平衡，将部分水替换成NA+后，每个单层蛋白质盖成电中性，然后按照这里的步骤按步骤进行。
制作alpha-A链结构
gmx pdb2gmx -f chain_A.pdb -o chain_A.gro -p chain_A.top -i chain_A.itp -water spc -ignh
生成相关文件后，用制作一个仿真盒子
gmx editconf -f chain_A.gro -o chain_A_box.gro -c -d -1.0 -bt cubic
用水溶解
gmx solvate -cp chain_A_box.gro -cs spc216.gro -p chain_A.top -o chain_A_water.gro
然后做离子平衡
gmx grompp -f ions.mdp -c chain_A_water -p chain_A.top -o chain_A_ions.tpr
这里我观察到系统成-16电荷，所以加16个NA+离子
gmx genion -s chain_A_ions.tpr -o chain_A_ions.gro -p chain_A.top -pname NA -np 16
ions.mdp文件如下
ions.mdp - used as input into grompp to generate ions.tpr
; Parameters describing what to do, when to stop and what to saveintegrator = steep
; Algorithm (steep = steepest descent minimization)emtol = 1000.0
; Stop minimization when the maximum force < 1000.0 kJ/mol/nmemstep = 0.01
; Minimization step sizensteps = 50000
; Maximum number of (minimization) steps to perform; Parameters describing how to find the neighbors of each atom and how to calculate the interactionsnstlist = 1
; Frequency to update the neighbor list and long range forcescutoff-scheme = Verlet
; Buffered neighbor searching ns_type = grid
; Method to determine neighbor list (simple, grid)coulombtype = cutoff
; Treatment of long range electrostatic interactionsrcoulomb = 1.0
; Short-range electrostatic cut-offrvdw = 1.0
; Short-range Van der Waals cut-offpbc = xyz
; Periodic Boundary Conditions in all 3 dimensions
然后打开vmd看一下图形
效果是正确的，可以继续进行。
然后就进行能量最小化
先放上em.mdp
; Run Control
integrator = steep ; A steepest descent algorithm for energy minimization
emtol = 100 ; Stop minimization when the maximum force < 100.0 kJ/mol/nm
emstep = 0.01 ; [nm] initial step-size
nsteps = 50000 ; Maximum number of (minimization) steps to perform
; Output Control
nstlog = 1 ; # of steps that else between writing energies to the log file
nstenergy = 1 ; # of steps that else between writing energies to energy file
; Neighbor searching and nonbonded interaction
cutoff-scheme = verlet ; Cutoff-related parameters
nstlist = 1 ; Frequency to update the neighbor list and the long-range forces
ns_type = grid ; Method to determine neighbor list (simple, grid)
pbc = xyz ; Use periodic boundary conditions in all directions
rlist = 0.5 ; [nm] Cut-off for making neighbor list (short range forces
; Electrostatics
coulombtype = PME ; Treatment of long range electrostatic interactions
rcoulomb = 0.5 ; [nm] Short-range electrostatic cut-off
; Van der waals
vdwtype = cutoff
rvdw = 0.5 ; [nm] Short-range Van der Waals cut-off
; Temperature and pressure coupling are off during EM
tcoupl = no ; No temperature coupling
pcoupl = no ; No pressure coupling (fixed box size)
; No velocities during EM
gen_vel = no ; Do not generate velocities
; options for bonds
constraints = none ; No contraints except those defined in the topology
这其中rlist,rcoulomb, rvdw,这几个值需要根据蛋白链的体积调整，gromacs会提示增加增加box size or 减少rlist，对了如果只减少这些值不行的话需要调整gro文件中box size，我都调整成2了。
gmx grompp -f em.mdp -c chain_A_ions.gro -p chain_A.top -o chain_A_ions_em.tpr
gmx mdrun -v -deffnm chain_A_ions_em
然后再打开看一下
看来也是正确的，没有出现类似聚集成一团或者空洞或者管状的结构。
制作beta-B链结构
制作通过同alpha-A链，只是参数不同，这里不再重复了。
制作最终结构
首先要做个水层，这里只有参数不同，不再赘述，详细过程同这篇
我们最终得到的文件是
gmx editconf -f small_water_npt.gro -o small_water_npt_center
然后就要添加蛋白层了
首先加下层
gmx insert-molecules -f small_water_npt_center.gro -ip position.dat -ci chain_A_ions_em.gro -o down2.gro -rot none
这个-rot none一定要加，否则结构不正确
然后先居中一下
gmx editconf -f down2.gro -o down2.gro
然后加上层
gmx insert-molecules -f down2.gro -ip position2.dat -ci chain_B_ions_em.gro -o down3.gro -rot none
两个position是有特定要求的，太远了达不到目的，太近了无法添加进去
position.dat
0 0 -4.8
position2.dat
0 0 2.1
这样就得到了最终的gro文件
下面我们还要制作top和ndx文件。
制作top
首先要先制作两个itp文件
由于内容过多，这里只叙述原理，就是把chain-A.top和chain-B.top中的[moleculetype] [atoms]等保留，而删除top文件中描述系统分子数量的语句，具体资源上传到这里了。
然后自己制作down3.top
#include "oplsaa.ff/forcefield.itp"
#include "oplsaa.ff/spce.itp"
#include "oplsaa.ff/ions.itp"
;#include "oplsaa.ff/spc.itp"
#include "chain_A_ions_em.itp"
#include "chain_B_ions_em.itp"
[ system ]
; Name
system
[ molecules ]
; Compound #mols
SOL 1487
Protein_chain_A 1
SOL 1401
NA 16
Protein_chain_B 1
SOL 1862
NA 15
这其中[ molecules ]的内容是分别从上面3个top中复制来的。
制作索引文件
使用gromacs的make_ndx制作索引文件
gmx make_ndx -f down3.gro -o down3.ndx
随后根据gromacs的提示依次选择
system
name
q
这样就完成索引文件的制作了。
尝试nvt预平衡
gmx grompp -f nvt1.mdp -c down3.gro -p down3.top -n down3.ndx -o fd_nvt.tpr
这条代码是可以成功的，说明理论上gromacs是可以做这个工作的
nvt1.mdp文件如下
; Run Control
integrator = md ; leap-frog integrator of Newton's equations of motion
tinit = 0 ; [ps] starting time for the run
dt = 0.002 ; 2 fs - [ps] time step for integration
nsteps = 5000 ; 0.1 ns
; Output Control
nstxout = 1000 ; save coordinates every 2 ps
nstenergy = 500 ; save energies every 1 ps
nstlog = 50000 ; update log file every 100 ps
; Bond parameters
continuation = no ; apply constraints to the start configuration and reset shells
constraints = h-bonds ; convert all bonds to constraints
constraint-algorithm = lincs ; holonomic constraints
; Neighbor searching and nonbonded interactions
cutoff-scheme = verlet ; Cutoff-related parameters
nstlist = 20 ; Frequency to update the neighbor list and the long-range forces
ns_type = grid ; Method to determine neighbor list (simple, grid)
pbc = xy ; Use periodic boundary conditions in all directions
rlist = 1.2 ; [nm] Cut-off for making neighbor list (short range forces)
ewald-geometry = 3dc
nwall = 2
wall-atomtype = MW MW
wall-type = 12-6
; Electrostatics
coulombtype = PME ; Treatment of long range electrostatic interactions
rcoulomb = 1 ; [nm] Short-range electrostatic cut-off
; Van der waals
vdwtype = cutoff
rvdw = 1 ; [nm] Short-range Van der Waals cut-off
; Do I need pme-order and fourier spacing?
;pme_order = 4 ; cubic interpolation
;fourierspacing = 0.16 ; grid spacing for FFT
; Temperature coupling
tcoupl = v-rescale ; Temperature coupling, modified Berendsen thermostat
tc_grps = system ; Group to couple to separate temperature baths
tau_t = 0.1 ; [ps] Time constant for coupling
ref_t = 300 ; [K] Reference temperature for coupling
; Pressure coupling is off for NVT
Pcoupl = No ; No pressure coupling (fixed box size)
; Generate velocities to start
gen_vel = yes ; assign velocities from Maxwell distribution
gen_temp = 300 ; temperature for Maxwell distribution
gen_seed = -1 ; generate a random seed
; Freeze Sheets
freezegrps = Backbone ;freezing the protein, so limit the water ;
freezedim = Y Y Y
但是这个还有个问题就是目前会报错
Step 0, time 0 (ps) LINCS WARNING
relative constraint deviation after LINCS:
rms 0.140735, max 4.433387 (between atoms 16877 and 16880)
bonds that rotated more than 30 degrees:
atom 1 atom 2 angle previous, current, constraint length
段错误 (核心已转储)
这个错误可以通过调整dt来解决。