Difference between revisions of "Sequence-based mutation analysis BCKDHA"
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− | | 29 || G||E|| ||0.37||w<font color=green>c</font>mP<font color=blue>d</font>IG<font color=green>nq</font><font color=red>Rh</font>V<font color=green>T</font><font color=red>k</font><font color=blue>e</font><font color=green>S</font>FLA<font color=green>y</font>|| |
+ | | 29 || G||E|| ||0.37||w<font color=green>c</font>mP<font color=blue>d</font>IG<font color=green>nq</font><font color=red>Rh</font>V<font color=green>T</font><font color=red>k</font><font color=blue>e</font><font color=green>S</font>FLA<font color=green>y</font>||[[File:BCKDHA_29.PNG]] |
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Revision as of 17:49, 23 June 2011
Contents
General
A Protocol was created where all steps for running the programs etc are described.
Subset of mutations
Reference amino acid | Mutated amino acid | Secondary Structure | |||||
---|---|---|---|---|---|---|---|
Position | Residue | Properties | Structure | Residue | Properties | Structure | |
29 | G | tiny, small | E | charged, polar | C | ||
125 | Q | acidic, polar | E | charged, polar | C | ||
166 | Y | hydrophobic, aromatic, polar | N | acidic, polar, small | H | ||
249 | G | tiny, small | S | polar, small, tiny, hydroxylic | H | ||
264 | C | sulphur containing, hydrophobic, tiny, small, polar | W | hydrophobic, aromatic, polar | E | ||
265 | R | charged, positive (basic), polar | W | hydrophobic, aromatic, polar | E | ||
326 | I | aliphatic, hydrophobic | T | hydroxylic, hydrophobic, small, polar | E | ||
361 | I | aliphatic, hydrophobic | V | aliphatic, hydrophobic, small | H | ||
409 | F | aromatic, hydrophobic | C | sulphur containing, hydrophobic, tiny, small, polar | C | ||
438 | Y | hydrophobic, aromatic, polar | N | acidic, polar, small | C |
Annotation: H = helix, E = beta-sheet, C = coil
To visualize the mutations in the three-dimensional protein structure, the PDB entry for BCKDHA, 1U5B, was used. As the PDB file only contains coordinate information about the protein itself, the signal peptide (45 first amino acids) are not annotated. Therefore the first mutation on position 29, which lies in the signal peptide, could not be visualized.
The Protocol describes in detail the way how we used pymol to visualize our mutations.
Position | AA1/AA2 | BLOSUM62 | PAM1 | PAM250 | |||
---|---|---|---|---|---|---|---|
score | worst | score | worst | score | worst | ||
29 | G/E | -2 | -4 (I, L) | 7 | 0 (I, W, Y) | 9 | 2 (W) |
125 | Q/E | 2 | -3 (C, F, I) | 27 | 0 (F, W, Y) | 7 | 1 (C, F, W) |
166 | Y/N | -2 | -3 (D, G, P) | 3 | 0 (R, D, Q, G, K, M, P) | 2 | 1 (A, R, D, Q, E, G, K, P) |
249 | G/S | 0 | -4 (I, L) | 21 | 0 (I, W, Y) | 11 | 2 (W) |
264 | C/W | -2 | -4 (E) | 0 | 0 (N, D, Q, E, G, L, K, M, F, W) | 1 | 1 (R, N, D, Q, E, L, K, M, F, W) |
265 | R/W | -3 | -3 (W, V, F, I, C) | 8 | 0 (D, E, G, Y) | 7 | 1 (F) |
326 | I/T | -1 | -4 (G) | 7 | 0 (G, A, P, W) | 4 | 1 (W) |
361 | I/V | 3 | -4 (G) | 33 | 0 (G, H, P, W) | 9 | 1 (W) |
409 | F/C | -2 | -4 (P) | 0 | 0 (D, C, Q, E, K, P, V) | 1 | 1 (R, D, C, Q, E, G, K, P) |
438 | Y/N | -2 | -3 (D, G, P) | 3 | 0 (R, D, Q, G, K, M, P) | 2 | 1 (A, R, D, Q, E, G, K, P) |
PSSM
In order to get a human-readable PSSM-File PsiBlast was run using the following command: blastpgp -i sequence.fasta -j 5 -d /data/blast/nr/nr -C profile.ckp -u 1 -J T
Multiple Sequence Alignment
To find the homologue sequences to BCKDHA we used BLAST. It found 250 homologous sequences, 25 of them are mammals.
ID | Accession | Entry name |
---|---|---|
sp | P11178 | ODBA_BOVIN |
sp | P12694 | ODBA_HUMAN |
sp | Q8HXY4 | ODBA_MACFA |
sp | P50136 | ODBA_MOUSE |
sp | A5A6H9 | ODBA_PANTR |
sp | P11960 | ODBA_RAT |
tr | Q6ZSA3 | Q6ZSA3_HUMAN |
tr | E7ESE6 | E7ESE6_HUMAN |
tr | B2R8A9 | B2R8A9_HUMAN |
tr | Q658P7 | Q658P7_HUMAN |
tr | E7EW46 | E7EW46_HUMAN |
tr | B4DP47 | B4DP47_HUMAN |
tr | Q59EI3 | Q59EI3_HUMAN |
tr | F1N5F2 | F1N5F2_BOVIN |
tr | B1PK12 | B1PK12_PIG |
tr | E2RPW4 | E2RPW4_CANFA |
tr | B2LSM3 | B2LSM3_SHEEP |
tr | F1RHA0 | F1RHA0_PIG |
tr | F1PI86 | F1PI86_CANFA |
tr | D2HMT3 | D2HMT3_AILME |
tr | Q2TBT9 | Q2TBT9_BOVIN |
tr | Q3U3J1 | Q3U3J1_MOUSE |
tr | Q99L69 | Q99L69_MOUSE |
tr | Q5EB89 | Q5EB89_RAT |
tr | B1WBN3 | B1WBN3_RAT |
With this 25 results we made a multiple alignment by using CLUSTALW. The alignment with all mammalian homologous was quite bad because of the sequences "Q6ZSA3" and "E2RPW4". These two sequences are much longer than the other one. So we removed those sequences and realigned the other sequences.
SNAP
To run SNAP we used the command:
snapfun -i BCKDHA.fasta -m mutations.txt -o SNAP.out
nsSNP | Prediction | Reliability Index | Expected Accuracy |
---|---|---|---|
G29E | Neutral | 0 | 53% |
Q125E | Non-neutral | 1 | 63% |
Y166N | Non-neutral | 2 | 70% |
G249S | Neutral | 1 | 60% |
C264W | Non-neutral | 4 | 82% |
R265W | Non-neutral | 4 | 82% |
I326T | Non-neutral | 3 | 78% |
I361V | Neutral | 4 | 85% |
F409C | Non-neutral | 4 | 82% |
Y438N | Non-neutral | 4 | 82% |
A second SNAP run was performed where all ten chosen mutation positions were mutated by all possible substitutions.
SIFT
Position | Reference AA | Mutated AA | SIFT prediction | SIFT Matrix | Prediction | ||
---|---|---|---|---|---|---|---|
Predict Not Tolerated | Seq Rep | Predict Tolerated | |||||
29 | G | E | 0.37 | wcmPdIGnqRhVTkeSFLAy | |||
125 | Q | E | y w v t s r p n m l kihgfedc a | 0.98 | Q | ||
166 | Y | N | cp<onft color=blue>dmeqkngrtisval | 1.00 | FHYW | ||
249 | G | S | |||||
264 | C | W | |||||
265 | R | W | |||||
326 | I | T | hdwpn<font color=blue<eqc</fontrsgkytaM | 1.00 | FLVI | File:BCKDHA 326 | |
361 | I | V | hwdgnqryekspcfmtlA | 1.00 | VI | ||
409 | F | C | |||||
438 | Y | N |
Polyphen2
Position | AA1/AA2 | HumDiv | HumVar | ||||||
---|---|---|---|---|---|---|---|---|---|
prediction | Score | Sensitivity | Specificity | prediction | Score | Sensitivity | Specificity | ||
29 | G/E | benign | 0.025 | 0.96 | 0.80 | benign | 0.018 | 0.96 | 0.52 |
125 | Q/E | possibly damaging | 0.759 | 0.85 | 0.93 | benign | 0.285 | 0.87 | 0.75 |
166 | Y/N | probably damaging | 0.997 | 0.40 | 0.98 | probably damaging | 0.964 | 0.59 | 0.93 |
249 | G/S | benign | 0.145 | 0.93 | 0.86 | benign | 0.292 | 0.86 | 0.75 |
264 | C/W | probably damaging | 1.000 | 0.00 | 1.00 | probably damaging | 1.000 | 0.00 | 1.00 |
265 | R/W | probably damaging | 1.000 | 0.00 | 1.00 | probably damaging | 1.000 | 0.00 | 1.00 |
326 | I/T | probably damaging | 0.997 | 0.40 | 0.98 | probably damaging | 0.998 | 0.16 | 0.99 |
361 | I/V | benign | 0.039 | 0.95 | 0.82 | benign | 0.178 | 0.89 | 0.70 |
409 | F/C | probably damaging | 0.998 | 0.27 | 0.99 | probably damaging | 0.939 | 0.64 | 0.92 |
438 | Y/N | probably damaging | 1.000 | 0.00 | 1.00 | probably damaging | 0.987 | 0.49 | 0.96 |
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