Structure-based mutation analysis GLA
by Benjamin Drexler and Fabian Grandke
Contents
Introduction
In this task we analyse the structure of our protein to find out what effects the point mutations have. Therefor we created mutated structures and compared them to the wild-type protein. Several tools based on different methods have been used to achieve that aim. We used the mutations that we have chosen in the previous task.
Methods
In the first step of this task we had to find available protein structures for our Protein and to decide which one would be the best for our detailed analysis. We set several cut-offs to exclude improper structures.
SCWRL
SCWRL was initially developed by Dunbrack et al. in 1997. We use SCWRL4<ref name=dunb>G. G. Krivov, M. V. Shapovalov, and R. L. Dunbrack, Jr. Improved prediction of protein side-chain conformations with SCWRL4. Proteins (2009)</ref> which was published in 2009. The program takes a PDB file and a sequence file as input. By usage of a rotamer library, collision detection, and a residue interaction graph the optimal side-chain conformation is calculated, based on the backbone and the mutated sequence given in the input files. The output is a PDB file containing the conformation and the total minimal energy of the graph in STDOUT.
FoldX
FoldX was developed by Serrano et al. in 2002<ref name=serr>Guerois R, Nielsen JE, Serrano L., Predicting Changes in the Stability of Proteins and Protein Complexes: A Study of More Than 1000 Mutation. Journal of Molecular Biology (2002)</ref>. We used version FoldX 3.0 beta 4. The program provides the calculation of determination of energy effects of point mutations. It provides different run modes, but basically it takes a PDB file as input calculates several single energies(e.g. Van der Waals, Electrostatics, ...) and returns the single energies together with the total energy as output.
Minimise
Before this tool from the virtual box was used we had to remove the hydrogens and waters from the PDB file with the script repairPDB. Afterwards we were able to compare the energies differences between the wildtype and the mutated protein.
GROMACS
Results
Structure Selection
There are several structure files available for our protein:
| PDB ID | Resolution [Å] | ph-Value | R-Factor | Coverage [%] | Missing Residues |
|---|---|---|---|---|---|
| 1R46 | 3.25 | 8.0 | 0.262 | 99.7 | 422-429 |
| 1R47 | 3.45 | 8.0 | 0.285 | 99.5 | 422-429 |
| 3GXN | 3.01 | NULL | 0.239 | 88.08 | 422-429 |
| 3GXP | 2.20 | NULL | 0.204 | 81.9 | 422-429 |
| 3GXT | 2.70 | NULL | 0.245 | 97.29 | 422-429 |
| 3HG2 | 2.30 | 4.6 | 0.178 | 97.32 | 422-429 |
| 3HG3 | 1.90 | 6.5 | 0.167 | 98.64 | 427-435 |
| 3HG4 | 2.30 | 4.6 | 0.166 | 99.86 | 422-429 |
| 3HG5 | 2.30 | 4.6 | 0.192 | 100 | 422-429 |
| 3LX9 | 2.04 | 6.5 | 0.178 | 98.92 | 423-435 |
| 3LXA | 3.04 | 6.5 | 0.216 | 99.52 | 427-435 |
| 3LXB | 2.85 | 6.5 | 0.227 | 99.3 | 427-435 |
| 3LXC | 2.35 | 6.5 | 0.186 | 98.31 | 423-435 |
We set two cutoffs to decide which structures are excluded:
- ph-value: < 6.5
- resolution: > 2.7
After we applied the cutoffs to our set of structures three were left (exclusion factors are colored red in the table). One of them was slightly better than the other ones so we decided to use 3HG3 (worse values are colored gray in the table). Additionally 3GH3 has the best overall resolution and R-factor (colored green). As the missing residues are very similar for all structures they are not further taken into account.
Mutation Mapping
Energy Comparison
| Number | AA-Position | Codon change | Amino acid change | SCWRL4 | FoldX | FoldX - Diff | Minimise | Minimise - Diff |
|---|---|---|---|---|---|---|---|---|
| WT | - | -20.93 | - | -20481.23 | - | |||
| 1 | 42 | ATG-ACG | Met -> Thr | 343.25 | 157.29 | -178.22 | -20324.41 | -156.82 |
| 2 | 65 | AGT-ACG | Ser -> Thr | 327.798 | 152.87 | -173.8 | -20339.34 | -141.89 |
| 3 | 117 | ATT-AGT | Ile -> Ser | 333.027 | 157.97 | -178.9 | -20353.47 | -127.76 |
| 4 | 143 | cGCA-ACA | Ala -> Thr | 333.944 | 154.40 | -175.33 | -20339.32 | -141.91 |
| 5 | 186 | CAC-CGC | His -> Arg | 323.717 | 154.57 | -175.5 | -20321.32 | -159.91 |
| 6 | 205 | gCCT-ACT | Pro -> Thr | 340.619 | 155.96 | -176.89 | -20345.87 | -135.36 |
| 7 | 244 | gGAC-CAC | Asp -> His | 333.594 | 152.08 | -173.01 | -20393.12 | -88.11 |
| 8 | 283 | CAG-CCG | Gln -> Pro | 332.631 | 159.91 | -180.84 | -8027.71 | -12453.52 |
| 8.2 | - | - | - | - | - | - | -19134.48 | -1346,95 |
| 9 | 321 | tCAG-TAG | Gln -> Glu | 332.853 | 160.95 | -181.88 | -20246.98 | -234.25 |
| 10 | 363 | TATa-TAA | Arg -> Cys | 330.56 | 150.50 | -171.43 | -20295.77 | -185.46 |
Gromacs
Wildtype
| Force Field | Average | Error Estimat | RMSD | Tot-Drift (kJ/mol) |
|---|---|---|---|---|
| Bond | ||||
| AMBERGS | 1826.99 | 420 | 4409.39 | -2499.37 |
| AMBER03 | 1639.74 | 410 | 4358.68 | -2424.42 |
| CHARMM27 | 2908.14 | 350 | 4779.8 | -2033.44 |
| Angle | ||||
| AMBERGS | 5496.47 | 74 | 476.18 | 408.548 |
| AMBER03 | 5324.13 | 72 | 469.75 | 369.24 |
| CHARMM27 | 7975.2 | 86 | 798.12 | 432.901 |
| Potential | ||||
| AMBERGS | -114713 | 1200 | 5648.79 | -7915.46 |
| AMBER03 | -91307.7 | 1200 | 5559.82 | -7839.05 |
| CHARMM27 | 136.699 | 32 | 64.3892 | 227.896 |
Mutations
| Force Field | Average | Error Estimat | RMSD | Tot/Drift |
|---|---|---|---|---|
| Bond | ||||
| 1 | 1815.39 | 570 | 5166.85 | -3384.48 |
| 2 | 1862.77 | 610 | 5331.85 | -3618.04 |
| 3 | 1773.13 | 520 | 4937.34 | -3068.93 |
| 4 | 1828.63 | 580 | 5229.18 | -3479.09 |
| 5 | 1870.95 | 610 | 5361.67 | -3713.22 |
| 6 | 1816.6 | 550 | 5091.81 | -3303.34 |
| 7 | 1819.7 | 570 | 5173.34 | -3397.07 |
| 8 | 2992.15 | 1700 | -nan | -10631.8 |
| 9 | 2083.16 | 830 | -nan | -4913.82 |
| 10 | 1867.42 | 620 | 5390.82 | -3693.03 |
| Angle | ||||
| 1 | 5183.95 | 85 | 360.959 | 550.303 |
| 2 | 5195.33 | 80 | 364.473 | 515.645 |
| 3 | 5196.5 | 89 | 353.256 | 586.473 |
| 4 | 5175.59 | 85 | 364.496 | 547.465 |
| 5 | 5113.99 | 81 | 365.511 | 526.244 |
| 6 | 5200.44 | 85 | 356.964 | 553.934 |
| 7 | 5261.77 | 87 | 365.202 | 565.196 |
| 8 | 5178.73 | 76 | -nan | 215.036 |
| 9 | 5201.95 | 76 | -nan | 442.141 |
| 10 | 5174.48 | 88 | 375.775 | 555.294 |
| Potential | ||||
| 1 | -90528.4 | 1600 | 7234.09 | -10149.1 |
| 2 | -90481.9 | 1600 | 7442.03 | -10340 |
| 3 | -90654 | 1500 | 6928.73 | -9614.54 |
| 4 | -90541 | 1600 | 7311.04 | -10343.7 |
| 5 | -91011.7 | 1600 | 7484.45 | -10592.5 |
| 6 | -90782.2 | 1600 | 7226.99 | -10188.5 |
| 7 | -90232.9 | 1600 | 7236.24 | -10198 |
| 8 | -87316 | 3600 | -nan | -23670.3 |
| 9 | -90090.3 | 1900 | -nan | -12335.3 |
| 10 | -89721.8 | 1700 | 7523.88 | -10750.1 |
References
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