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Revision as of 18:00, 1 July 2011
Contents
Task description
A detailed task description can be found here.
Selection of protein structure
We had the following choice of reference structures for PAH:
| Entry | Method | Resolution (A) | Chain | Positions |
|---|---|---|---|---|
| 1DMW | X-Ray | 2.00 | A | 118-424 |
| 1J8T | X-Ray | 1.70 | A | 103-427 |
| 1J8U | X-Ray | 1.50 | A | 103-427 |
| 1KW0 | X-Ray | 2.50 | A | 103-427 |
| 1LRM | X-Ray | 2.10 | A | 103-427 |
| 1MMK | X-Ray | 2.00 | A | 103-427 |
| 1MMT | X-Ray | 2.00 | A | 103-427 |
| 1PAH | X-Ray | 2.00 | A | 117-424 |
| 1TDW | X-Ray | 2.10 | A | 117-424 |
| 1TG2 | X-Ray | 2.20 | A | 117-424 |
| 2PAH | X-Ray | 3.10 | A/B | 118-452 |
| 3PAH | X-Ray | 2.00 | A | 117-424 |
| 4PAH | X-Ray | 2.00 | A | 117-424 |
| 5PAH | X-Ray | 2.10 | A | 117-424 |
| 6PAH | X-Ray | 2.15 | A | 117-424 |
All these structures have in common that they did not solve the structure of the whole PAH protein. They only solve the catalytic domain of PAH, the missing parts are the tetramerisation domain and the regulatory domain which are located at the N- and C- terminal ends. In addition, there is no complete true apo structure available either. All structures have at least a Fe2+ atom bound. Because of this we thought it might be better if we select a structure which has all reaction components or at least most of them bound in the catalytic site in order to get a good picture of the binding site configuration. Though, only 1KW0 and 1MMK fulfilled the constrains that all reaction components are bound.
In the end we did not select 1KW0 or 1MMK, we decided us for the structure 1J8U which is complexed with Fe2+ and BH4 (5,6,7,8-TETRAHYDROBIOPTERIN). Only. This has simple reasons. First of all it has the lowest resolution (1.5 Angstrom) and secondly we already used this structure in previous task as our reference structure for PAH. So we think to keep our experiments more consistent we should stay with this structure. Furthermore, we identified this structure to have no gaps and it solves the complete catalytic domain (as all available structures). Also, the R-Value looked good to us which is 0.157.
To sum it up our selected structure 1J8U has the following experimental metrics (taken from PDBe):
Mapping mutations to the structure
We identifier the following functional residues and catalytic sites with the help of UniProt entry P00439 and Catalytic Site Atlas. We looked for catalytic sites in the structure of 1J8U.
We identified the following functional residues and catalytic sites:
- HIS 285, functional part: side chain (from CSA)
- HIS 290 (from UniProt)
- GLU 330 (from UniProt)
- SER 349, functional part: side chain (from CSA)
In the following picture we can see the Fe+2 atom as a brown sphere, BH4 as a cloud of green blue and red spheres, the location of the mutated residues in orange (mutation I65T and R71H are not included) and the four identified catalytic sites in yellow:
I65T
This mutation is not part of our structure but we would say probably no effect on catalytic site because it is too far away.
R71H
This mutation is not part of our structure but we would say probably no effect on catalytic site because it is too far away.
R158Q
Probably no effect on catalytic site because it is too far away.
R261Q
Probably no effect on catalytic site because it is too far away.
T266A
No direct influence on catalytic site residue. However, this residue is located what we would define as the catalytic center.
P275S
Probably no effect on catalytic site because it is too far away.
T278N
No direct influence on catalytic site residue. However, this residue is located what we would define as the catalytic center. ´
P281L
Probably direct influence on catalytic site residue HIS 285. In addition, this residue is located what we would define as the catalytic center.
G312D
Probably no effect on catalytic site because it is too far away.
R408W
Probably no effect on catalytic site because it is too far away.
Introducing mutations to 1J8U
Introducing mutations to 1J8U with SCWRL
We had to employ several steps to introduce our mutated residues to our structure with SCWRL:
1. extract amino acid sequence from PDB file
/apps/scripts/repairPDB 1J8U.pdb -seq > 1J8U_seq.txt
2. convert all upper case residues to lower case
vim 1J8U_seq.txt
:%s/.*/\L&/g
3. create one sequence file for each mutation and put the residue to mutate as an uppercase letter
4. execute SCWRL for each mutation
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_R158Q.txt -o 1J8U_R158Q.pdb | tee scwrl_r158q.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_R261Q.txt -o 1J8U_R261Q.pdb | tee scwrl_r261q.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_T266A.txt -o 1J8U_T266A.pdb | tee scwrl_t266a.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_P275S.txt -o 1J8U_P275S.pdb | tee scwrl_p275s.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_T278N.txt -o 1J8U_T278N.pdb | tee scwrl_t278n.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_P281L.txt -o 1J8U_P281L.pdb | tee scwrl_p281l.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_G312D.txt -o 1J8U_G312D.pdb | tee scwrl_g312d.out
/apps/scwrl4/Scwrl4 -i 1J8U.pdb -s 1J8U_seq_R408W.txt -o 1J8U_R408W.pdb | tee scwrl_r408w.out
SCRWL versus PyMol: Comparison of the rotation of the side chains
I65T
Could not compare the side chain of this mutation since this position is not included in 1J8U.
R71H
Could not compare the side chain of this mutation since this position is not included in 1J8U.
R158Q
R261Q
T266A
P275S
T278N
P281L
G312D
R408W
Minimise
| Mutation | Energy |
|---|---|
| I65T | - |
| R71H | - |
| R158Q | -7400.825142 |
| R261Q | -7456.793410 |
| T266A | -7392.572699 |
| P275S | -7418.432874 |
| T278N | -7379.215571 |
| P281L | -7401.621858 |
| G312D | -5643.645312 |
| R408W | -5438.301688 |
Gromacs
Mutations
| Mutation | Steps | Potential Energy | Maximum Force | Norm of Force |
|---|---|---|---|---|
| R158Q | 328 | -3.7326676e+04 | 5.1247034e+02 | 1.7521566e+01 |
| R261Q | 363 | -3.7302664e+04 | 1.4321185e+02 | 1.3723365e+01 |
| T266A | 315 | -3.7422707e+04 | 3.7855130e+02 | 1.6683250e+01 |
| P275S | 256 | -3.7569789e+04 | 3.6591925e+02 | 1.9855389e+01 |
| T278N | 272 | -3.7567461e+04 | 6.8363385e+02 | 2.0818382e+01 |
| P281L | 336 | -3.7656289e+04 | 1.6796155e+02 | 1.4324168e+01 |
| G312D | 334 | -3.7583254e+04 | 8.6771161e+02 | 2.3178673e+01 |
| R408W | 320 | -3.6683172e+04 | 6.0292523e+02 | 2.2048130e+01 |
I65T
Could not compare the side chain of this mutation since this position is not included in 1J8U.
R71H
Could not compare the side chain of this mutation since this position is not included in 1J8U.
R158Q
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 726.495 | 230 | 2165.47 | -1392.61 | 
|
| Angle | 2351.59 | 22 | 189.806 | 80.2662 | 
|
| Potential | -36202.9 | 740 | 3182.59 | -4873.47 | 
|
R261Q
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 705.135 | 210 | 2053.34 | -1228.48 | 
|
| Angle | 2356.01 | 21 | 179.696 | 95.0119 | 
|
| Potential | -36270.8 | 690 | 3020.89 | -4437.22 | 
|
T266A
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 740.335 | 240 | 2209.81 | -1459.51 | 
|
| Angle | 2352.51 | 22 | 192.08 | 65.6116 | 
|
| Potential | -36283.8 | 750 | 3239.42 | -4913.99 | 
|
P275S
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 911.556 | 410 | -nan | -2575.37 | 
|
| Angle | 2338.07 | 33 | -nan | -61.3735 | 
|
| Potential | -36106.6 | 1000 | -nan | -6645.9 | 
|
T278N
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 778.485 | 280 | 2383 | -1740.86 | 
|
| Angle | 2344.25 | 25 | 207.028 | 26.8598 | 
|
| Potential | -36291.6 | 820 | 3493.71 | -5504.87 | 
|
P281L
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 728.439 | 220 | 2141.57 | -1346.93 | 
|
| Angle | 2352.62 | 21 | 187.673 | 80.9581 | 
|
| Potential | -36549.7 | 740 | 3172.53 | -4826.89 | 
|
G312D
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 1074.93 | 550 | 3492.54 | -3394.39 | 
|
| Angle | 2396.13 | 28 | 198.398 | -68.8 | 
|
| Potential | -13546.2 | 23000 | 328802 | -143251 | 
|
R408W
| Category | Average | Err.Est. | RMSD | Tot-Drift | Graph |
| Bond | 1508.76 | 980 | 4742.06 | -5893.55 | 
|
| Angle | 2482.65 | 99 | 386.434 | -483.529 | 
|
| Potential | 4.48468e+07 | 4.3e+07 | 6.93881e+08 | -2.71572e+08 | 
|
Timerun
The calculation of the time runs could not be done by a script, therefore we ran gromacs for just AMBER03, AMBERGS and CHARMM each with the nsteps of 125, 250, 500 and 1000.
