Structure-based mutation analysis HEXA
Farbcode bei active side: active side: grün Glycolysation: gelb Cystein: cyan Mutation:rot
Contents
Sequence Description
We had to use a PDB file, in which are no missing residues and the quality of the structure should be high. We found only one PDB structure which was not bounded to a ligand. Therefore, we could not regard the quality and the pH value, the R-factor and the coverage. Nevertheless, we listed in the following table this values:
| experiment type | X-Ray diffraction |
| Resolution | 2.8 Å |
| temperature (Kelvin) | 100K |
| temperature (Celsius) | -173 °C |
| pH-Value | 5.5 (slightly acid) |
| R-Value | 0.270 |
It was not possible to find one file, without any missing residues. In each file there was a gap between residue 74 to 89 and the last amino acid. Therefore, we decided to cut off the first 89 residues and use a PDB file with a structure from 89 - 528. This file can be found [here].
Mutations
Because of the shorten PDB file, it was not possible for us to analyse the first two mutations on position 29 and 39.
| SNP-id | codon number | mutation codon | mutation triplet |
| rs4777505 | 29 | Asn -> Ser | AAC -> AGC |
| rs121907979 | 39 | Leu -> Arg | CTT -> CGT |
| rs61731240 | 179 | His -> Asp | CAT -> GAT |
| rs121907974 | 211 | Phe -> Ser | TTC -> TCC |
| rs61747114 | 248 | Leu -> Phe | CTT -> TTT |
| rs1054374 | 293 | Ser -> Ile | AGT -> ATT |
| rs121907967 | 329 | Trp -> TER | TGG -> TAG |
| rs1800430 | 399 | Asn -> Asp | AAC -> GAC |
| rs121907982 | 436 | Ile -> Val | ATA -> GTA |
| rs121907968 | 485 | Trp -> Arg | gTGG -> CGG |
Analysis of the mutations
We created for each mutation an extra page. The summary of the analysis can be seen in the Summary Section.
Protocoll - Using the methods
Pymol
We visualized the local hydrogen-bonding network with following commands:
distance hbonds, all, all, 3.2, mode=2 zoom resi <interval> hide labels, all color red, resi <mutation_position>
Furthermore, we also used the polar contact mode in pymol to visulize the h-bonds.
The clashed are visualized by the following commands:
distance clash = pos_mutation, all, 2.0, 0 zoom clash
FoldX
To use FoldX, we created a runfile, which can be found [here]. We fitted the temperature and pH-value to the values we extracted from the PDB page. Furthermore, we analysed the mutations with a random choosen temperature and pH value, to see how much influence these parameteres have on the analysis.
We ran FoldX with following command:
FoldX -runfile run.txt > foldx_output
minimise
Next we used minimise. Therefore, it was not necessary to create any file for the run. Sadly, we could not find any documentation about minimise and therefore, it was really hard to figure out how it works and what means the output.
We ran minimise with following command:
minimise <input> <output>
Gromacs
Before we could run Gromacs, we had to curate our PDB file. Therefore, we used the script repairPDB to extract chainA. Next we run SCRWL to make sure, that every residue is available in the PDB file.
Then we used the commands which are listed in our task section.
Additionally, to the analysis of our mutated sequences, we also chose different forcefields and analysed our protein without any mutation with these forcefields.
Here are the results of this analysis:
| analysed energies (in kJ/mol) | force field | |||
| AMBER3 | AMBER99SB-ILDN | CHARMM27 | ||
| Bond | Average | 852.968 | 1091.57 | 1796.6 |
| Err Estimation | - | 270 | 240 | |
| RMSD | 42.0241 | -nan | 2924.29 | |
| Drift | -74.0853 | -1622.75 | -1404.11 | |
| Angle | Average | 3438.47 | 3326.81 | 4764.7 |
| Error Estimation | - | 62 | 60 | |
| RMSD | 16.8864 | -nan | 466.82 | |
| Drift | -33.7041 | 404.076 | 368.45 | |
| Potential | Average | -50917.7 | -61304.1 | 166.582 |
| Error Estimation | - | 960 | 39 | |
| RMSD | 66.4149 | -nan | 79.7058 | |
| Drift | -132.636 | -6402.44 | 280.841 | |
Furthermore, we used different numbers for nsteps. The result of how long these analysis run, can be found in the following table and graph:
| nstep | time real | time user | time sys | #steps |
| 50 | 8.268s | 3.860s | 0.110s | 24 |
| 500 | 27.523s | 47.650s | 0.540s | 321 |
| 5000 | 25.281s | 17.710s | 0.210s | 114 |
| 50000 | 14.940s | 14.210s | 0.300s | 91 |
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