^{by Benjamin Drexler and Fabian Grandke}

Introduction

In this task we analysed the simulated data that have been created within task 8. We used several tools of GROMACS to analyse the data and Pymol to visualize them. The single steps were done according to the tutorial from the task 10 page.

Methods/Materials

Results

Brief check of the results

How many frames are in the trajectory file and what is the time resolution?

Wildtype	Mutation 3	Mutation 8
2001 frames	2001 frames	2001 frames
time resolution of 5ps	time resolution of 5ps	time resolution of 5ps

How long did the simulation run in real time (hours), what was the simulation speed (ns/day) and how many years would the simulation take to reach a second?

Wildtype	Mutation 3	Mutation 8
18h06:37	18h29:13	18h19:11
13.252 ns/day	12.982 ns/day	13.101 ns/day
~206740 years	~211040 years	~209123 years

Which contribution to the potential energy accounts for most of the calculations?

Wildtype	Mutation 3	Mutation 8
-8.52573e+05 kJ/mol	-8.53327e+05 kJ/mol	-8.52539e+05 kJ/mol

Visualization of the results

Wildtype	Mutation 3	Mutation 8
Figure 1: Molecular Dynamics simulation of the wildtype protein.	Figure 2: Molecular Dynamics simulation of the mutation 3 protein.	Figure 3: Molecular Dynamics simulation of the mutation 8 protein.

Figures 1,2 ,and 3 show every ~30 frame of the molecular dynamics simulation. An animated figure of all 1000 simulated frames would be to large for this wiki. All animations show similar but not identical movement of the protein.

Quality assurance

Convergence of energy terms

Temperature

Description	Wildtype	Mutation 3	Mutation 8
Max(kJ/mol)	301.3734	302.0039	301.7407
Min(kJ/mol)	293.9565	294.0463	293.9268
Average(kJ/mol)	297.9275	297.9489	297.9303
Plot	Figure 4: Temperature over time for wildtype.	Figure 5: Temperature over time for mutation 3.	Figure 6: Temperature over time for mutation 8.

The mutated proteins (Figures 5 and 6) temperatures averages are slightly higher than the wildtype proteins (Figure 4) one, but there is no significant increase. The lowest temperature of the mutation 8 protein is even lower than the minimum of the wildtype. The general appearance of the temperature diagram differs between the proteins, but there is no change into a certain direction.

Pressure

Description	Wildtype	Mutation 3	Mutation 8
Max(kJ/mol)	346.2387	385.5467	326.9994
Min(kJ/mol)	-308.7182	-357.4386	-291.1198
Average(kJ/mol)	0.4503482	-1.521002	-0.7279793
Plot	Figure 7: Pressure over time for wildtype.	Figure 8: Pressure over time for mutation 3.	Figure 9: Pressure over time for mutation 8.

Figures 7, 8 and 9 show the pressure values during the simulation. All proteins have high levels of variation (>600 kJ/mol) and have averages around zero. The mutated proteins averages both are slightly negative, but in the circumstances of the high overall variation this seems not significant.

Potential

Description	Wildtype	Mutation 3	Mutation 8
Max(kJ/mol)	-849914.5	-850058.5	-849804.2
Min(kJ/mol)	-855729.5	-856170.2	-855717.7
Average(kJ/mol)	-852568.2	-853314.1	-852547.7
Plot	Figure 10: Potential over time for wildtype.	Figure 11: Potential over time for mutation 3.	Figure 12: Potential over time for mutation 8.

Figures 10, 11 and 12 show the potential values during the simulation. Similar to the pressure values, there are high variations around a value of -853,000 and the simulations seem to have not reached the certain equilibria and there is no obvious difference between the wildtype protein and the mutated ones.

Total Energy

Description	Wildtype	Mutation 3	Mutation 8
Max(kJ/mol)	-695814.5	-696662.8	-695560
Min(kJ/mol)	-703550.4	-703769	-702829.8
Average(kJ/mol)	-699602.2	-700202.2	-699497.3
Plot	Figure 13: Total energy over time for wildtype.	Figure 14: Total energy over time for mutation 3.	Figure 15: Total energy over time for mutation 8.

Figures 13,14 and 15 show the values of the total energy during the simulation. All diagrams show variation around a value of ~700,000 and there are no results that show a reasonable difference between the proteins.

Minimum distances between periodic images

Description	Wildtype	Mutation 3	Mutation 8
Minimum distance(nm)	1.932	1.992	2.007
Time of occurance	370	7910	5940
Plot	Figure 16: Minimum distance between periodic boundary cells for wildtype.	Figure 17: Minimum distance between periodic boundary cells for mutation 3.	Figure 18: Minimum distance between periodic boundary cells for mutation 8.

Figures 16,17 and 18 show the minimum distance between periodic boundary cells for the certain proteins. The minimum distances of the mutated proteins are slightly higher than the wildtype protein ones. If the minimum distance would be smaller and be under a cutoff value, that would mean that there would be interactions of different parts of the molecule what would cause huge changes in molecular dynamics movement, and the results would be completely different.

Root mean square fluctuations

Wildtype	Mutation 3	Mutation 8
Figure 19: Root mean square fluctuations for wildtype protein.	Figure 20: Root mean square fluctuations for mutation 3 protein.	Figure 21: Root mean square fluctuations for mutation 8 protein.
Figure 22: Root mean square fluctuations for wildtype C-alpha.	Figure 23: Root mean square fluctuations for mutation 3 C-alpha.	Figure 24: Root mean square fluctuations for mutation 8 C-alpha.
Figure 25: Image of aligned average and b-factor protein for wildtype protein.	Figure 26: Image of aligned average and b-factor protein for mutation 3 protein.	Figure 27: Image of aligned average and b-factor protein for mutation 8 protein.
Figure 28: Image of aligned average and b-factor protein for wildtype C-alpha.	Figure 29: Image of aligned average and b-factor protein for mutation 3 C-alpha.	Figure 30: Image of aligned average and b-factor protein for mutation 8 C-alpha.

Figures 19, 20 and 21 show the RMS fluctuation of the proteins. The diagrams are scaled differently so, but the pattern of peaks are very similar. The only difference and the reason for the different scaling is the last upswing. It is most extreme in mutation 3 protein(>5) and smallest in mutation 8 protein (~2.5). The other values are almost the same. So again there is no obvious difference between the wildtype and the mutated proteins.

Figures 22, 23 and 24 show the RMS fluctuations of the proteins C-alphas. The results have the same character as the previous ones. Again there is only a difference in the last upswing.

Figures 25, 26 and 27 show alignments of the average proteins and the b-factor ones from pymol. They look very similar, as well. On the left side of the picture is a helix that "points" towards the viewer. It is the only part of the protein that is red colored in red. It is the same end of the protein that showed significant behavior in the two steps before. The other parts of the proteins are mostly color dark blue or light blue. The segments that are highlighted light blue are mostly similar for all proteins. Only the mutation 8 protein has some of those colored in green. That means that the b-factor values of those are a little bit higher. All three proteins align very well.

Figures 28, 29 and 30 show alignments of the average proteins C-alpha and the b-factor ones from pymol. Despite some small differences the alignments look very similar. Again only the end of the chain is colored significantly. The dark blue averages and light blue b-factor atoms align very well, again.

Convergence of radius of gyration

Wildtype	Mutation 3	Mutation 8
Figure 31: Radius of gyration over time for wildtype protein.	Figure 32: Radius of gyration over time for mutation 3 protein.	Figure 33: Radius of gyration over time for mutation 8 protein.

Figures 31, 32 and 33 show the radius of gyration of the proteins. The black line shows the general change of the proteins structure during the simulation. The green, red and blue lines show the certain values for the X-, Y- and Z-axis, respectively. The general structure seems not to change at all, despite by very little variation. The blue and red line seem to be anti proportional, every time one has an upswing, the other one has a downswing. They are not exactly mirrored, but the general tendency is very obvious. The green line is more independent. It behaves different in every protein and does not seem to influence the general change of the proteins structures.

Solvent accessible surface area

Description	Wildtype	Mutation 3	Mutation 8
SAS over time	Figure 34: Solvent accessible surface area over time for wildtype protein.	Figure 35: Solvent accessible surface area over time for mutation 3 protein.	Figure 36: Solvent accessible surface area over time for mutation 8 protein.
SAS per atom	Figure 37: Solvent accessible surface area per atom for wildtype protein.	Figure 38: Solvent accessible surface area per atom for mutation 3 protein.	Figure 39: Solvent accessible surface area per atom for mutation 8 protein.
SAS per residue	Figure 40: Solvent accessible surface area per residue for wildtype protein.	Figure 41: Solvent accessible surface area per residue for mutation 3 protein.	Figure 42: Solvent accessible surface area per residue for mutation 8 protein.

Figures 34, 35 and 36 show the solvent accessible surface area of the proteins. The black, red, green and blue lines show the hydrophobic, hydrophilic, total and D Gsolv, respectively. All diagrams show that the hydrophobic solvents accessibility is very slightly higher than the hydrophilic solvents one, but they are very close. The green lines indicate that the total energy stays at a constant level, despite a little variance. There is no significant difference between the three diagrams.

Figures 37, 38 and 39 show the solvent accessible surface area of the proteins per atom. The three diagrams look alike, but have some differences (e.g. Figure 39 does not have the significant double peak(>0.4) at atom ~400).

Figures 40, 41 and 42 show the solvent accessible surface area of the proteins per residue. Again, the diagrams are very similar, but there are significant changes at the positions where the mutations occur. In Figure 41 at residue 117 the value is 0.34, so there is an increase in comparison to the wildtype value that is 0.18. In Figure 42 at residue 283 the value increases from 0.022 to 0.11(wildtype).

Hydrogen bonds

Description	Wildtype	Mutation 3	Mutation 8
Internal HB	Figure 43: Internal hydrogen bonds over time for wildtype protein.	Figure 44: Internal hydrogen bonds over time for mutation 3 protein.	Figure 45: Internal hydrogen bonds over time for mutation 8 protein.
Protein-Solvent HB	Figure 46: Hydrogen bonds between protein and surrounding solvents for wildtype protein.	Figure 47: Hydrogen bonds between protein and surrounding solvents for mutation 3 protein.	Figure 48: Hydrogen bonds between protein and surrounding solvents for mutation 8 protein.

Figures 43, 44 and 45 show the number of internal hydrogen bonds within the proteins. The diagrams are very similar and show almost no variation. They have the same value of about 350 internal hydrogen bonds. That number does not significantly change over time.

Figures 46, 47 and 48 show the number of hydrogen bonds between the protein and surrounding solvents. Again, the diagrams look alike, but there is more variation within the lines. The number of hydrogen bonds during the simulation lies between the 650 and 720. The mutations do not seem to influence the number of hydrogen bonds.

Ramachandran (phi/psi) plots

General Ramachandran	Wildtype	Mutation 3	Mutation 8
Figure 49: General ramachandran plot.<ref name=rama_wiki>http://en.wikipedia.org/wiki/Ramachandran_plot</ref>	Figure 50: Internal hydrogen bonds over time for wildtype protein.	Figure 51: Internal hydrogen bonds over time for mutation 3 protein.	Figure 52: Internal hydrogen bonds over time for mutation 8 protein.

Analysis of dynamics and time-averaged properties

Root mean square deviations again

Description	Wildtype	Mutation 3	Mutation 8
Plot	Figure 53: RMSD matrix for wildtype protein.	Figure 54: RMSD matrix for mutation 3 protein.	Figure 55: RMSD matrix for mutation 8 protein.

Cluster analysis

Description	Wildtype	Mutation 3	Mutation 8
Plot	Figure 56: RMS distribution for wildtype protein.	Figure 57: RMS distribution for mutation 3 protein.	Figure 58: RMS distribution for mutation 8 protein.
Plot	Figure 59: Image of the largest two clusters of wildtype protein.	Figure 60: Image of the largest two clusters of mutation 3 protein.	Figure 61: Image of the largest two clusters of mutation 8 protein.