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Results Analysis.md

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Results Analysis

0. Check the results you get

  • module load gromacs/2018.3
  • gmx check -f step7_1.trr

1. Concatenates several input trajectory files in sorted order

  • gmx trjcat -f step7_1.trr -settime -o step7.trr

*For -settime, use c option

2. Convert trajectory files to smaller trajectory file for viewer

2.1 Name a new group of protein-ligand complex and create a new index file using gmx make_ndx if necessary

Launch gmx make_ndx

  • gmx make_ndx -f step6.6_equilibration.gro -o protein-ligand.ndx

Combine group 1 and 13 to get new group 18: Protein-NNR, and then q to save/export new index file "protein-ligand.ndx"

GROMACS:      gmx make_ndx, version 2018.3
Executable:   /usr/local/apps/gromacs/2018.3/bin/gmx
Data prefix:  /usr/local/apps/gromacs/2018.3
Working dir:  /gpfs/gsfs10/users/dout2/PnuC_3NR-XY100-charmm-gui-0226106078/gromacs
Command line:
  gmx make_ndx -f step6.6_equilibration.gro -o protein-ligand.ndx


Reading structure file
Going to read 0 old index file(s)
Analysing residue names:
There are:   707    Protein residues
There are: 19664      Other residues
Analysing Protein...
Analysing residues not classified as Protein/DNA/RNA/Water and splitting into groups...

  0 System              : 96403 atoms
  1 Protein             : 11490 atoms
  2 Protein-H           :  5677 atoms
  3 C-alpha             :   707 atoms
  4 Backbone            :  2121 atoms
  5 MainChain           :  2825 atoms
  6 MainChain+Cb        :  3483 atoms
  7 MainChain+H         :  3517 atoms
  8 SideChain           :  7973 atoms
  9 SideChain-H         :  2852 atoms
 10 Prot-Masses         : 11490 atoms
 11 non-Protein         : 84913 atoms
 12 Other               : 84913 atoms
 13 NNR                 :    99 atoms
 14 POPC                : 26666 atoms
 15 SOD                 :    52 atoms
 16 CLA                 :    67 atoms
 17 TIP3                : 58029 atoms

 nr : group      '!': not  'name' nr name   'splitch' nr    Enter: list groups
 'a': atom       '&': and  'del' nr         'splitres' nr   'l': list residues
 't': atom type  '|': or   'keep' nr        'splitat' nr    'h': help
 'r': residue              'res' nr         'chain' char
 "name": group             'case': case sensitive           'q': save and quit
 'ri': residue index

> 1 | 13

Copied index group 1 'Protein'
Copied index group 13 'NNR'
Merged two groups with OR: 11490 99 -> 11589

 18 Protein_NNR         : 11589 atoms

>

2.2 Then, load the new index file and center the protein-ligand complex

  • gmx trjconv -f step7_1.trr -s step7_1.tpr -n protein-ligand.ndx -o PnuC_3NR_500ns.trr -pbc cluster -center -dt 1000

*Select group 18 "Protein_NNR" for clustering, group 18 "Protein_NNR" for centering, and the whole system (group 0) for output.

Otherwise, we can also center the protein directly

  • gmx trjconv -f step7_1.trr -s step7_1.tpr -o PnuC_3NR_500ns.trr -pbc cluster -dt 1000 -center

  • gmx trjconv -f step7_1.trr -s step7_1.tpr -o PnuC_3NR_500ns.trr -pbc cluster -dt 1000 -center -ur compact

  • gmx trjconv -f step7_1.trr -s step7_1.tpr -o PnuC_3NR_500ns.trr -pbc whole -dt 1000 -center

  • gmx trjconv -f step7_1.trr -s step7_1.tpr -o PnuC_3NR_500ns.trr -pbc mol -dt 1000 -center -ur compact

  • gmx trjconv -f step7_1.trr -s step7_1.tpr -o PnuC_3NR_500ns.trr -pbc nojump -dt 1000 -center

*Choose “Protein” for cluster, "Protein" to center, and “whole system” for output

#How to load vmd on my Linux and view the trajectory file

  • cd /home/dout2/Application/vmd_bin
  • ./vmd

Convert .trr to .pdb file for Pymol view

  • gmx trjconv -f step7.trr -s step6.0_minimization.tpr -o PnuC_0NR_1us.pdb -pbc nojump -dt 1000

*Choose “Protein” for output

3. Quality Assurance

Whether the protein is stable and close to the experimental structure.

3.1 Convergence of Energy Terms Extraction of some thermodynamic parameters from the energy file: temperature, pressure, potential energy, kinetic energy, unite cell volume, density, and the box dimensions, etc. Energy analysis:

  • gmx eneconv -f step7_1.edr -o PnuC_0NR_1us.edr -settime
  • gmx energy -f PnuC_0NR_1us.edr -o temperature.xvg
  • xmgrace temperature.xvg Pressure:
  • gmx energy -f PnuC_0NR_1us.edr -o pressure.xvg
*choose pressure in the list
  • xmgrace pressure.xvg Energy:
  • gmx energy -f PnuC_0NR_1us.edr -o energy.xvg
*choose 16 “total energy” in the list
  • xmgrace energy.xvg Volume:
  • gmx energy -f PnuC_0NR_1us.edr -o volume.xvg
  • xmgrace volume.xvg Density:
  • gmx energy -f PnuC_0NR_1us.edr -o density.xvg
  • xmgrace density.xvg Box:
  • gmx energy -f PnuC_0NR_1us.edr -o box.xvg
  • xmgrace box.xvg

3.2 Minimum distances between periodic images gmx mindist -f step7.trr -s step6.0_minimization.tpr -od minimal-periodic-distance.xvg -pi

3.3 RMSF: Root mean square flections

  • gmx rmsf -f step7.trr -s step6.0_minimization.tpr -o rmsf-per-residue.xvg -ox average.pdb -res 
*select
  • xmgrace rmsf-per-residue.xvg

3.4 RMSD: Convergence of RMSD

  • gmx rms -f step7.trr -s step6.0_minimization.tpr -o rmsd-all-atom-vs-start.xvg 
*select protein-H
  • gmx rms -f step7.trr -s step6.0_minimization.tpr -o rmsd-all-backbone-vs-start.xvg 
*select only backbone atoms
  • echo 1 | gmx trjconv -f step7.trr -s step6.0_minimization.tpr -o peptide.trr 
*Check the convergence towards the average structure.
  • gmx rms -f step7.trr -s average.pdb -o rmsd-all-atom-vs-average.xvg
  • gmx rms -f step7.trr -s average.pdb -o rmsd-backbone-vs-average.xvg
  • xmgrace xx.xvg

Convert .xvg file to .png, for show and publication?

  • xmgrace -nxy rmsd-all-backbone-vs-start.xvg -hdevice PNG -hardcopy -printfile rmsd-all-backbone-vs-start.png

3.5 Convergence of Radius of Gyration

  • gmx gyration -f step7.trr -s step6.0_minimization.tpr -p -o radius-of-gyration.xvg
  • xmgrace radius-of-gyration.xvg

4. Structural Analysis: Properties derived from configurations

4.1 Hydrogen bonds

  • gmx hbond -f step7.trr -s step6.0_minimization.tpr -num hydrogen-bonds-intra-protein.xvg
  • gmx hbond -f step7.trr -s step6.0_minimization.tpr -num hydrogen-bonds-protein-other.xvg

4.2 Salt Bridge

4.3 Secondary Structure How to load DSSP:

  • module load DSSP
  • whereis dssp
  • export DSSP=/usr/local/apps/DSSP/2.2.1/bin/dssp
  • gmx do_dssp -f step7.trr -s step6.0_minimization.tpr -ver 2 -o secondary-structure.xpm -sc secondary-structure.xvg -dt 10
  • gmx xpm2ps -f secondary-structure.xpm -o secondary-structure.eps
  • display secondary-structure.eps 4.4 Ramachandran Plots
  • gmx rama -f step7.trr -s step6.0_minimization.tpr -o ramachandran.xvg
  • xmgrace ramachandran.xvg

5. Analysis of Dynamics and Time-Averaged properties

5.1 RMSD again

  • gmx rms -s step6.0_minimization.tpr -f step7.trr -f2 step7.trr -m rmsd-matrix.xpm -dt 10
  • gmx xpm2ps -f rmsd-matrix.xpm -o rmsd-matrix.eps
  • display rmsd-matrix.eps

5.2 How to generate a color coded RMSF using PyMol?

  • Action->Presets->B-Factor Putty

#Assign color by B-factor

  • B-factor coloring can be done with the spectrum command. Example:

spectrum b, blue_white_red, minimum=20, maximum=50 as cartoon cartoon putty

#Generate .pdb file by Gromacs

  • mx rmsf -f step7.trr -s step6.0_minimization.tpr -oq PnuC_0NR_1us_Bfactor.pdb

#Biowulf sbatch jobscript

#!/bin/bash

#rmsf2Bfactor.jobscript

#This script is to submit a sbatch job on Biowulf to calculate and generate a .pdb file with Bfactor inserted.

module load gromacs/2018.3

#Calculate using whole protein echo 1 | gmx rmsf -f step7.trr -s step6.0_minimization.tpr -oq PnuC_0NR_1us_Bfactor.pdb

#Calculate using backbone echo 4 | gmx rmsf -f step7.trr -s step6.0_minimization.tpr -oq PnuC_0NR_1us_Bfactor.pdb

5.3 Principal Components Analysis (PCA)

#Reference: #https://www3.mpibpc.mpg.de/groups/de_groot/compbio1/p4/index.html #http://scc.acad.bg/ncsa/downloads/programs/GROMACS/GROMACS%204.5%20Tutorial.pdf #http://thegrantlab.org/bio3d/tutorials/principal-component-analysis

#calculates and diagonalizes the (mass-weighted) covariance matrix #Select group “4” (Protein backbone) both for fit and analysis.

  • gmx covar -f step7.trr -s step6.0_minimization.tpr -o eigenval.xvg –v eigenvect.trr –xpma covara.xpm

#Use xpm2ps to make a pretty plot of the atomic covariance matrix.

  • gmx xpm2ps -f covara.xpm -o covara.eps -do covara.m2p

#Use ghostview (or Photoshop) to view the plot (gv covara.eps).

  • gv covara.eps

#To view the most dominant mode (1), use the following command ...

  • gmx anaeig -v eigenvect.trr -f step7.trr -s step6.0_minimization.tpr -first 1 -last 1 -nframes 100 -extr fws-ev1.pdb

#How to perfomr PCA using VMD

http://prody.csb.pitt.edu/tutorials/nmwiz_tutorial/pca.html

https://www.ks.uiuc.edu/Research/vmd/plugins/nmwiz/

5.4 Energy landscape