Structure-based mutation analysis
Contents
General
According to the UniProt entry about HFE_HUMAN are three 3D-structures of the HFE_HUMAN available, which are listed below. We have chosen the '1A6Z' because it has the best resolution, a very good R-Value (it measures the quality of the model obtained from the crystallographic data), a pH near the physiological optimum and is as good as complete. '1DE4' has a slightly better R-Value and pH, but this PDB also includes the transferrin receptor, which we do not need and do not want in our structure. Also the missing residues of chain A are the same as in the structure '1A6Z' which are only the first three positions. '1C42' is only a hypothetical model, so we exclude it from further research.
All stereochemistrical properties of the structure are shown in the figure to the right<ref>Lebrón JA, Bennett MJ, Vaughn DE, Chirino AJ, Snow PM, Mintier GA, Feder JN, Bjorkman PJ.: Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor.</ref>.
| PDB | Method | Resolution (Å) | Chain | R-Value | R-Free | pH | Temperature | Completeness | Missing residues (Chain:pos) | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1A6Z | X-ray | 2.60 | A/C | 0.233 | 0.277 | 6.5 | -150.15°C (123 k) | 98.0 | A:1-3 | C:1-3 | - | - | - | - |
| 1C42 | model | - | A | - | - | - | - | - | - | - | - | - | - | - |
| 1DE4 | X-ray | 2.80 | A/D/G | 0.231 | 0.265 | 8.0 | -173.15°C (100 k) | 94.3 | A:1-3 | C:121,757-760 | D:1-3 | F:121,757-760 | G:1-3 | I:121,757-760 |
We used the following 7 mutations, because we were not able to map 3 of them to the 1A6ZA chain mostly because of position errors. We are using the same names as given in Task 6. Mutation 8 had to be split into two parts because of possible mutation into to different amino acids.
| Mutation |
|---|
| Mutation 2 [M35T] |
| Mutation 3 [S65C] |
| Mutation 4 [I105T] |
| Mutation 5 [Q127H] |
| Mutation 6 [A176V] |
| Mutation 7 [T217I] |
| Mutation 8a [C282Y] |
| Mutation 8b [C282S] |
Mapping
Because we have no annotation about the active site so we just visualized the mutations at the '1A6Z' structure. Secondly we are using the same mutations as in Task 6 and have therefore the same problemes with their visualization (only 7 of 10 visualizeable).
All mutations are scattered accross the protein with no affinity to some special region or secondary structure element. Mutations shown in red are near glycosylation positions and mutations in yellow are near disulfidbonds.
Energy comparison
SCWRL
SCWRL predicts protein side-chain confirmations given a fixed backbone. We are using SCWRL version 4 released in 2009.
Usage:
- use only chain A of backbone pdb:
1A6ZA.pdb - extract amino acid sequence and change it to lowercase:
aa.txt - introduce each mutation into on seperated
aa_x.txtfile as capital- cmd:
scwrl -i 1A6ZA.pdb -s aa_x.txt -o ./mutant_pdbs/1A6ZA_mutant_x.pdb > 1A6ZA_mutant_x.txt
- cmd:
Results:
Robert: currently working.
| Mutation | Position | Energy | Energy normalized |
|---|---|---|---|
| Reference | -- | 247.944 | 1 |
| Mutation 2 [M35T] | 35 | 252.324 | 1,017665279 |
| Mutation 3 [S65C] | 65 | 246.695 | 0,994962572 |
| Mutation 4 [I105T] | 105 | 250.833 | 1,011651825 |
| Mutation 5 [Q127H] | 127 | 252.368 | 1,017842739 |
| Mutation 6 [A176V] | 176 | 280.381 | 1,130823896 |
| Mutation 7 [T217I] | 217 | 260.189 | 1,049386152 |
| Mutation 8a [C282Y] | 282 | 389.539 | 1,571076533 |
| Mutation 8b [C282S] | 282 | 255.859 | 1,031922531 |
- The energy is normalized by the wild-type structure. A value larger then 1 means that the energy is increased compared to the wild-type. A value smaller 1 shows a decreased energy.
Minimise
Minimise is able to minimise the energy of an model.
Usage:
- remove all hydrogen and water atoms from the pdb files with repairPDB:
1A6ZA_mutant_x_clean.pdb- cmd:
repairPDB 1A6ZA_mutant_x.pdb -nosol > ./repair_pdb/1A6ZA_mutant_x_clean.pdb
- cmd:
- minimise the energy of the models:
- cmd:
minimise 1A6ZA_mutant_x_clean.pdb ./minimised_pdb/1A6ZA_mutant_x_clean_minimised.pdb > 1A6ZA_mutant_x_clean_minimised.txt
- cmd:
Results:
Robert: currently working.
| Mutation | Position | Energy | Energy normalized |
|---|---|---|---|
| Reference | -- | -3724.153777 | 1 |
| Mutation 2 [M35T] | 35 | -5020.465319 | 1,348082174 |
| Mutation 3 [S65C] | 65 | -5040.815685 | 1,353546601 |
| Mutation 4 [I105T] | 105 | -5028.869826 | 1,35033893 |
| Mutation 5 [Q127H] | 127 | -5031.137220 | 1,350947765 |
| Mutation 6 [A176V] | 176 | -4957.946411 | 1,331294761 |
| Mutation 7 [T217I] | 217 | -5037.718631 | 1,352714988 |
| Mutation 8a [C282Y] | 282 | -2596.778899 | 0,697280256 |
| Mutation 8b [C282S] | 282 | -5017.057355 | 1,347167076 |
- The energy is normalized by the wild-type structure. A value larger then 1 means that the energy is increased compared to the wild-type. A value smaller 1 shows a decreased energy.
FoldX
FoldX scores the importance of amino acid interactions according to the overall stability of the protein and calculates the energy.
Usage:
- create a runfile tutorial and adjust all default parameters to known (if possible):
runfile.txt - create a listfile of all pdb files that should be included in energy calculation:
listfile.txt - run foldx with runlist
- cmd:
sudo ./foldx -runfile runfile.txt > output.txt
- cmd:
Results:
remark: somehow the foldx command crashed my virtual maschine and now it is broken. i need to get a new one o.O
Our licence has expired. We will do that after getting a new licence.
Gromacs
Has to be done!
References
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