Difference between revisions of "Sequence Alignments BCKDHA"
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=== Functionally important residues === |
=== Functionally important residues === |
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| − | The functionally important sites are according to [http://www.uniprot.org/uniprot/P12694] the following sites: |
+ | The functionally important sites are according to Uniprot[http://www.uniprot.org/uniprot/P12694] the following sites: |
| − | * Metal binding site, 206 |
+ | * Metal binding site, S: 206 (161) |
| − | * Metal binding site, 211 |
+ | * Metal binding site, Q: 211 (166) |
| − | * Metal binding site, 212 |
+ | * Metal binding site, I: 212 (167) |
| + | As the Uniprot sequence is 445 aa long and the PDB sequence (1U5B) only 400 aa (without transit sequence), one has to consider the offset. The functional site positions in brackets are used to determine their conservation in the multiple sequence alignments. |
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| + | |||
| + | [[File:BCKDHA_clustalW.PNG|right|100px]] |
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| + | [[File:BCKDHA_cobalt.PNG|right|100px]] |
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| + | [[File:BCKDHA_muscle.PNG|right|100px]] |
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| + | [[File:BCKDHA_TCoffee.PNG|right|100px]] |
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| + | [[File:BCKDHA_TCoffee3D.PNG|right|100px]] |
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=== References === |
=== References === |
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Revision as of 19:14, 23 May 2011
Contents
Sequence Alignments
Sequence searches
- FASTA
../bin/fasta36 sequence.fasta database > FastaOutput.txt
- BLAST
blastall -p blastp -d database -i sequence.fasta > BlastOutput.txt
- PSIBLAST
blastpgp -i sequence.fasta -j iterations -h evalueCutoff -d database > PsiblastOutput.txt
- HHSearch
hhsearch -i query -d database -o output.txt
database = /data/blast/nr/nr
Result Statistics
- Overlap
To illustrate the overlap of the returned sequences for the sequences searches, Venn Diagrams were drawn.
The PSI-BLAST runs with each 3 iterations returned absolutely the same sequences match our query BCKDHA. The same is true for the PSI-BLAST runs with each 5 iterations. This fact was used to combine their results for the following Venn Diagramm (created with [6]):
Including BLAST and FASTA, the most interesting fact is that FASTA found more than 2600 more results, that were not returned by any of the other search algorithms. This may be due to no restriction concerning the E-value using the default FASTA search. BLAST returned only one additional Hit, that was not found in any of the PSI-BLAST searches, but which is also included in the FASTA results. Any PSI-BLAST results were also detected by FASTA. Using PSIBLAST with 5 iterations, 6 more Hits were returned than using PSIBLAST with only 3 iterations. This may be due to a longer search time. All in all one could say, that the Search algorithms returned nearly the same sequences matching our query.
Only HHSearch returned not only quite few aligned sequences (10), but also sequences, that were not found by any of the other algorithms. The corresponding Venn Diagram is shown below.
- Identity Distribution
As the PSIBlast runs with each 3 iterations resulted in the same Hits, as well as the PSI-BLAST runs with 5 iterations, the identity distributions for those runs were pooled using the same colour. Remarkable is the identity distribution for the FASTA run, which returned a lot more hits with little identity, that the other runs. All in all, FASTA returned almost 3000 Hits (default parameter search), while all the BLAST/PSIBLAST runs returned not more than 300 Hits each. Therefore a lot of the FASTA 'Hits' have little identity and a quite low E-value (see below), but still FASTA returned some good results.
- Evalue Distribution
The E-value distribution for the sequence searches is quite similar concerining the BLAST and PSI-BLAST results. Only FASTA has a lot wider E-value range, which can be explained by the fact, that FASTA returned about 10 times more Hits, among which a lot of sequences have little identity and therefore a quite high E-value.
- HSSP recall
The comparison with the HSSP alignment for our protein BCKDHA posed another challenge: HSSP alignment files use Swissprot/EMBL Identifier which had to be mapped to GI numbers to be comparable to our sequence files. The mapping was performed using the ID Mapping tool provided by NCBI[7]. The 3350 Swissprot Identifiers were mapped to almost 6000 GI numbers, but still about 500 identifiers could not be mapped at all. Doing the mapping the other way round and converting all GI numbers from the BLAST and FASTA runs, the same problem arised. For the recall evaluation the second mapping was chosen to display and the corresponding plot can be seen below.
As expected from the large number of Hits in the HSSP file, most of the listed related proteins were not identified by the other alignment tools. The best overlap can be observed with PSIBLAST (5 iterations, but the same is true for the 3 iteration runs and BLAST, as their outputs are nearly identical), where 90% of the converted IDs are also returned by HSSP. A large fraction of FASTA is also covered in HSSP. But as FASTA was run without any E-value restriction it returned also a lot of sequences with low identity which are not likely to have the same structure and are therefore not found using the structural alignment tool HSSP.
Afterwards the overlap of sequences for the different alignment methods were compared to the HSSP sequences.
Sequences chosen for the multiple Alignment:
| SeqIdentifier | Seq Identity | source |
|---|---|---|
| 99-90% Sequence Identity | ||
| 56967006|pdb|1X7Z | 99% | PSI BLAST, 3 iterations, E-value cutoff 0.005 |
| 7546384|pdb|1DTW | 95% | BLAST |
| 34810149|pdb|1OLU | 99% | PSI BLAST, 3 iterations, E-value cutoff 10E-6 |
| 13277798|gb|AAH03787.1 | 95% | PSI BLAST, 3 iterations, E-value cutoff 10E-6 |
| 148727347|ref|NP_001092034.1 | 95% | BLAST |
| 89-60% Sequence Identity | ||
| 196011048|ref|XP_002115388.1 | 66% | PSI BLAST, 3 iterations, E-value cutoff 0.005 |
| 149543950|ref|XP_001517857.1 | 67% | BLAST |
| 47227873|emb|CAG09036.1 | 82,5% | FASTA |
| 47196273|emb|CAF88112.1 | 81% | PSI BLAST, 5 iterations, E-value cutoff 0.005 |
| 12964598|dbj|BAB32665.1 | 88% | PSI BLAST, 5 iterations, E-value cutoff 10E-6 |
| 59-40% Sequence Identity | ||
| 193290664|gb|ACF17640.1 | 47% | BLAST |
| 215431443|ref|ZP_03429362.1 | 40% | FASTA |
| 225557347|gb|EEH05633.1 | 51% | PSI BLAST, 3 iterations, E-value cutoff 10E-6 |
| 58267618|ref|XP_570965.1 | 50% | PSI BLAST, 5 iterations, E-value cutoff 0.005 |
| 162449842|ref|YP_001612209.1 | 41% | PSI BLAST, 5 iterations, E-value cutoff 10E-6 |
| 39-20% Sequence Identity | ||
| 56966700|pdb|1W85 | 31% | PSI BLAST, 3 iterations, E-value cutoff 0.005 |
| 5822330|pdb|1QS0 | 38.1% | FASTA |
| 13516864|dbj|BAB40585.1 | 33% | PSI BLAST, 3 iterations, E-value cutoff 10E-6 |
| 284166853|ref|YP_003405132.1 | 35% | PSI BLAST, 5 iterations, E-value cutoff 0.005 |
| 76800932|ref|YP_325940.1 | 34% | PSI BLAST, 5 iterations, E-value cutoff 10E-6 |
Sequences for the Multiple Sequences Alignment were downloaded via NCBI, the sequence id can be changed in the link to retrieve the fasta format: http://www.ncbi.nlm.nih.gov/protein/76800932?report=fasta
Multiple Alignments
clustalw sequences.fasta
t_coffee -seq sequences.fasta
t_coffee -seq sequences.fasta -mode expresso
muscle -in sequences.fasta -out output.aln
download cobalt
./cobalt -i sequences.fasta -norps T > output.aln
Conservation and Gaps
| Alignment methods | Gaps | Conserved Columns | ||||||
|---|---|---|---|---|---|---|---|---|
| Gaps | Avg Gap Length | 100% cons | >90% cons | >80% cons | >70% cons | >60% cons | >50% cons | |
| ClustalW | 12 | 3,75 | 24 | 50 | 31 | 49 | 54 | 72 |
| T-Coffee | 25 | 4,56 | 24 | 50 | 31 | 49 | 54 | 72 |
| T-Coffee (3D) | 56 | 4,75 | 21 | 45 | 34 | 49 | 64 | 71 |
| Cobalt | 19 | 3,26 | 24 | 55 | 31 | 45 | 60 | 71 |
| Muscle | 17 | 4,76 | 26 | 46 | 22 | 31 | 14 | 8 |
Gaps in secondary structure
ClustalW
| Gap position | Gap length | Secondary structure |
| 109-110 | 4 | Helix |
| 142-143 | 1 | Helix |
| 235-236 | 1 | Beta strand |
| 276-277 | 11 | Helix |
| 294-295 | 1 | Beta strand |
| 394-395 | 5 | Helix |
T-Coffee
| Gap position | Gap length | Secondary structure |
| 141-142 | 1 | Helix |
| 232-233 | 1 | Beta strand |
| 275-276 | 11 | Helix |
| 310-311 | 1 | Helix |
| 369-370 | 5 | Turn |
| 395-396 | 18 | Helix |
| 398-399 | 5 | Helix |
T-Coffee 3d
| Gap position | Gap length | Secondary structure |
| 101-102 | 1 | Helix |
| 108-109 | 4 | Helix |
| 115-116 | 1 | Helix |
| 116-117 | 1 | Helix |
| 141-142 | 1 | Helix |
| 153-154 | 1 | Beta strand |
| 163-164 | 1 | Helix |
| 177-178 | 3 | Helix |
| 234-235 | 1 | Beta strand |
| 263-264 | 4 | Beta strand |
| 265-266 | 1 | Beta strand |
| 276-277 | 2 | Helix |
| 308-309 | 8 | Helix |
| 309-310 | 5 | Helix |
| 314-315 | 6 | Helix |
| 362-363 | 6 | Helix |
| 371-372 | 4 | Turn |
| 376-377 | 1 | Helix |
| 380-381 | 1 | Helix |
| 382-383 | 7 | Helix |
| 383-384 | 3 | Helix |
| 384-385 | 2 | Helix |
| 387-388 | 2 | Helix |
| 394-395 | 5 | Helix |
Cobalt
| Gap position | Gap length | Secondary structure |
| 108-109 | 4 | Helix |
| 141-142 | 1 | Helix |
| 177-178 | 3 | Helix |
| 276-277 | 11 | Helix |
| 294-295 | 1 | Beta strand |
| 305-306 | 1 | Helix |
| 311-312 | 2 | Helix |
| 387-388 | 1 | Helix |
| 388-389 | 1 | Helix |
| 395-396 | 13 | Helix |
Muscle
| Gap position | Gap length | Secondary structure |
| 109-110 | 4 | Helix |
| 141-142 | 1 | Helix |
| 177-178 | 3 | Helix |
| 276-277 | 11 | Helix |
| 294-295 | 1 | Beta strand |
| 305-306 | 1 | Helix |
| 394-395 | 5 | Helix |
Functionally important residues
The functionally important sites are according to Uniprot[8] the following sites:
- Metal binding site, S: 206 (161)
- Metal binding site, Q: 211 (166)
- Metal binding site, I: 212 (167)
As the Uniprot sequence is 445 aa long and the PDB sequence (1U5B) only 400 aa (without transit sequence), one has to consider the offset. The functional site positions in brackets are used to determine their conservation in the multiple sequence alignments.
References
Secondary structure information
back to Reference Sequence of BCKDHA
back to Maple syrup urine disease main page
