Canavan Task 2 - Sequence alignments
Contents
Sequence Search
Sorry, guys, we're a bit behind schedule! Hope to have everything finished before 11pm tonight (Monday) and hope that's early enough for you to read. Sorry again! Susi and Fanny
Meh, take your time, not like we aren't busy with our introductory talk ;) - jonas
Sequence
The native ASPA sequence that we used for the current task is shown below:
UniProt: P45381
>hsa:443 ASPA, ACY2, ASP; aspartoacylase; K01437 aspartoacylase [EC:3.5.1.15] (A)
MTSCHIAEEHIQKVAIFGGTHGNELTGVFLVKHWLENGAEIQRTGLEVKPFITNPRAVKK
CTRYIDCDLNRIFDLENLGKKMSEDLPYEVRRAQEINHLFGPKDSEDSYDIIFDLHNTTS
NMGCTLILEDSRNNFLIQMFHYIKTSLAPLPCYVYLIEHPSLKYATTRSIAKYPVGIEVG
PQPQGVLRADILDQMRKMIKHALDFIHHFNEGKEFPPCAIEVYKIIEKVDYPRDENGEIA
AIIHPNLQDQDWKPLHPGDPMFLTLDGKTIPLGGDCTVYPVFVNEAAYYEKKEAFAKTTK
LTLNAKSIRCCLH
Search
BLASTP
We ran BlastP on student machines with the big_80 as a reference database.
Command:
blastall -p blastp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o blastp_p45381_wt_big80.out
| Parameters | default E-Value = 10 | E-Value 10e-10 |
| results | 196 | 94 |
| best E-Value | 1e-155 | 1e-155 |
| worst E-Value | 9.6 | e-15 |
| comment | Most of the resulting proteins are Aspartoacylases of other species. Most of the results with EValue > e-15 are Succinylglutamate Desuccinylases, which catalyze a reaction similar to Aspartoacylase. | The results are the same as for the first run, just with an earlier cutoff |
PSIBLAST
PSIBlast was used in the same fashion as BLAST, with the big_80 as the background database. Commands:
- Running 2 iterations and default E-Value 0.002
blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it2_p45381_wt_big80.out -j 2
- 2 iterations, more strict E-value cutoff of 10E-10
blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it2_h10e10_p45381_wt_big80.out -j 2 -h 10e-10
- 10 iterations, default Evalue 0.002
blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it10_p45381_wt_big80.out -j 10
- 10 iterations, E-value cutoff 10E-10
blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it10_h10e10_p45381_wt_big80.out -j 10 -h 10e-10
| Parameters | it2, def E-Value (0.002) | it2 E-Value 10e-10 | it10 def E-Value (0.002) | it10 E-Value 10e-10 |
| time | ~2m30 | ~2m30 | ~10m | time: ~10m |
| results | 500 | 93 | 500 | 500 |
| best E-Value | 1e-142 | 1e-145 | 5e-70 | 7e-70 |
| worst E-Value | 3e-4 | 2e-29 | 8e-38 | 1e-38 |
| comments | Results with best EValues are mostly Aspartoacylases, Sequences previously not found are mostly Succinylglutamate Desuccinylases | results mainly Aspartoacylases | - converged after 8 rounds - most significant results include more Succinylglutamate Desuccinylases than Aspartoacylases | - all 10 iterations were done (no early convergence) - aspartoacylases slightly more frequent in lower E-Values (< E-58), but no significant difference in E-Values for aspas and succis |
HHBLITS
Run HHBlits on student machines with Uniprot20 database.
Commands:
- 2 iterations:
hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -o hhblits_p45381_def.out
- 8 iterations:
hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -n 8 -o hhblits_p45381_n10.out
-n number of iterations (def 2)
| Parameters | it 2 | it 8 |
| time | 2m50 | ~6m |
| results | 274 | 500 |
| best E-Value | 2e-110 | 2.9e-68 |
| worst E-Value | 0.0011 | 9.5e-09 |
| comment | mixed results with Aspartoacylases and Succi | very varying results: Aspartoacylasen, Succinylasen, Zinc Proteins |
Summary and Comparison
Along with the expactations one can find more hits with Psi-Blast than with a simple Blast search.
In general, one can distinguish between two kinds of proteins, that frequently are identified by the sequence searches:
- Aspartoacylases
- Succinylglutamate Desuccinylases
BlastP
A simple blast search yields only about 90 significant hits if one considers a threshold of 10e-10 as a significance cutoff. As one can see in Figure ??, the restriction of the E-Value results in less hits with a low sequence similarity.
Psi Blast
Increasing the amount of iterations performed in a PSI-Blast search, obviously increases the running time. One can see, that the best ranked hits of the runs with 10 iterations have lower E-Values than the best hits of the runs with less iterations. Yet, the result includes a larger amount of significant hits with higher E-Values. This means, increasing the iterations finds further distantly related sequences, which is the expected outcome. This outcome is also represented in the distribution of sequence identities. As one can see in figure ??, running PSI-Blast with 10 iterations results in hits with a lower sequence identity to our query sequence than the hits from the run with 2 iterations.
When restricting the E-Value Cutoff for the profile built-up, we found that more hits are classified as Aspartoacylases than as Succinylglutamate Desuccinylases. The running time, as well as the E-Values of the resulting hits did not change significantly. The majority of the results from the runs with only two iterations, has moderate sequence identities with a broad distribution between 10% and 50%. In contrast, the results from the run with 10 iterations split up into two groups of hits which form cluster at about 15% and 35% sequence identity. This difference is also represented in the E_Value distribution. The runs with 10 iterations result in Hits with moderate E_Values between -200 and -40 log(E_Values). The runs with 10 iterations in contrast result in many low significant hits (log(E_Value > -20)) and a variety of high significant hits.
HHBlits
Running HHBlits with 2 iterations yields a small amount of hits (270) with very low (2e-110) and very high (0.0011) E-Values. To increase the amount of hits, we repeated the HHBlits search with the maximum amount of 8 iterations which resulted in a broader output with more Hits with lower averaged E-Values (compare figure ??). Regarding the Sequence Identity distribution, running HHBlits with 8 iterations results in more distant related Hits (see Figure ??).
Overlap
As one can see in Figure ??, roughly 40 percent of the resulting hits are unique to each method. From our considerations, about 25 percent of the hits are significant hits, that could be further investigated (overlap of 50 percent).
Further Evaluation
We tried to further validate the sequence search hits via structural similarity. Unfortunately none of the resulting Hits was a PDB Hit. Furthermore we tried to map the sequence identifiers against the UniProtKB/Swiss-Prot PDB cross-references (http://www.uniprot.org/docs/pdbtosp.txt). Again, this mapping yielded no results, which is why we cannot include any structural information for our ongoing research. When inspecting the annotation for the sequence hits, we already found, that the majority of the hits codes for Aspartoacylases or respectively the highly related protein Succinylglutamate Desuccinylases. Since there already exists a crystal structure of the human Aspartoacylase, it is only reasonable that one will not find other structures for this class of proteins. Additionally, a huge amount of hits codes for not yet characterized proteins, which also will hardly be an interesting target for crystallization.
Multiple Sequence Alignments
For generating our dataset for the MSA we clustered all Hits into Sequence Identity groups:
- >90%: 1
- 60-89%: 59
- 40-59%: 197
- 20-39%: 1141
Since we only got one hit with an sequence Identity >90% we decided to group out hits as follows: three groups of sequences with eight members each:
- 60-99%
- 40-59%
- 20-39%
We chose those hits from the respective groups, that have been found by at least 4 methods (overlap of 50%).
id eVal identity coverage alignment_length # whole range = Set100 tr|C7PCU7|C7PCU7_CHIPD 5e-63 21 0.9933 474 tr|B3RSE1|B3RSE1_TRIAD 2e-93 49 0.8415 362 tr|G2PG26|G2PG26_STRVO 1e-105 33 0.5854 427 tr|Q8RX86|Q8RX86_ARATH 1e-105 35 0.9814 422 tr|G8NYA7|G8NYA7_GRAMM 4e-61 22 0.6186 470 tr|H1Q7I8|H1Q7I8_9ACTO 1e-97 37 0.5409 396 tr|E1ZHK5|E1ZHK5_CHLVA 8e-80 38 0.8485 368 sp|Q0CEF5|AGALG_ASPTN 4e-63 12 0.611 478 tr|F5BFS9|F5BFS9_TOBAC 1e-106 36 0.9534 410 tr|F8FLU8|F8FLU8_PAEMK 1e-76 10 0.6091 474 # <40% sequence identity = Set40 tr|B8P149|B8P149_POSPM 3e-80 28 0.9425 432 tr|G2TQE8|G2TQE8_BACCO 7e-68 8 0.5795 452 tr|F9HJT9|F9HJT9_9STRE 3e-70 11 0.5709 452 tr|H2JN17|H2JN17_STRHY 3e-69 23 0.774 504 tr|C5AKH4|C5AKH4_BURGB 2e-67 26 0.5488 403 sp|Q0CEF5|AGALG_ASPTN 4e-63 12 0.611 478 tr|B3CFN7|B3CFN7_9BACE 1e-78 26 0.5828 412 tr|D4KDQ2|D4KDQ2_9FIRM 8e-76 10 0.611 483 tr|D4W2N5|D4W2N5_9FIRM 1e-67 10 0.5478 435 tr|F2USV1|F2USV1_SALS5 1e-88 35 1 467 # >60% sequence identity = Set60 tr|G1P280|G1P280_MYOLU 1e-108 78 0.9699 420 tr|Q4RTE7|Q4RTE7_TETNG 7e-89 71 0.7319 314 tr|F1Q5G5|F1Q5G5_DANRE 1e-106 67 0.9138 392 tr|E1B725|E1B725_BOVIN 1e-111 76 0.9727 428 tr|H2U095|H2U095_TAKRU 1e-101 65 0.9424 412 tr|G1T044|G1T044_RABIT 1e-109 82 0.9698 417 tr|C0HA45|C0HA45_SALSA 1e-102 63 0.9534 409 tr|H0WQ54|H0WQ54_OTOGA 4e-87 71 0.9953 428 tr|G3WK18|G3WK18_SARHA 1e-108 72 0.9388 414 tr|H2L5H7|H2L5H7_ORYLA 1e-100 61 0.9534 411
ClustalW
command: clustalw -align -infile=./db_over60.fa -outfile=./clustalw_msa_60.aln
In the alignment of the >60% group the first two motifs are not colored in the alignment. This is due to two very short sequences which produce gaps in the alignment and thus lower the consensus.
TCoffee
Muscle
When comparing the three MSAs one can identify some conserved regions. Especially the two groups with sequence identities <60% show very similar MSAs.
There are three strongly conserved motivs:
- GGTHGNE
- DLNR
- DLHNT
In the alignment of the >60% group the first two motifs are not colored in the alignment. This is due to two very short sequences which produce gaps in the alignment and thus lower the consensus.
