Difference between revisions of "Canavan Task 2 - Sequence alignments"

From Bioinformatikpedia
(BLASTP)
(BLASTP)
Line 10: Line 10:
   
   
<figtable id="aspa_blastp">
 
 
<table border = 0>
 
<table border = 0>
 
<tr>
 
<tr>
Line 19: Line 18:
 
<td>
 
<td>
   
  +
  +
<figtable id="aspa_blastp">
 
<table cellspacing=0 align="center" cellpadding=5>
 
<table cellspacing=0 align="center" cellpadding=5>
 
<caption align="center"><b><xr nolink id="aspa_blastp"/></b> <br> Results from a BlastP search with Aspartoacylse using different EValues.</caption>
 
<caption align="center"><b><xr nolink id="aspa_blastp"/></b> <br> Results from a BlastP search with Aspartoacylse using different EValues.</caption>

Revision as of 09:06, 24 August 2012

Protocol

Further information can be found in the protocol.


Pairwise Sequence Search

BLASTP

Even when using the non-restricitve default EValue cutoff, we only find 196 hits. Using a stricter cutoff, we find 94 significant hits. Most of the resulting proteins are Aspartoacylases of other species. Most of the results with EValue > e-15 are Succinylglutamate Desuccinylases, which are in the same protein family (Desuccinylase / Aspartoacylase family) and catalyze a reaction similar to Aspartoacylase.


<figtable id="aspa_blastp">

<xr nolink id="aspa_blastp"/>
Results from a BlastP search with Aspartoacylse using different EValues.
EValue default (10) 10e-10
results19694
best E-Value1e-1551e-155
worst E-Value9.6e-15

</figtable>

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-Value1e-142 1e-145 5e-70 7e-70
worst E-Value3e-4 2e-29 8e-38 1e-38
commentsResults with best EValues are mostly Aspartoacylases, Sequences previously not found are mostly Succinylglutamate Desuccinylasesresults 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)

Parametersit 2 it 8
time2m50~6m
results274500
best E-Value2e-1102.9e-68
worst E-Value0.00119.5e-09
commentmixed results with Aspartoacylases and Succivery 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.

Comparison of distribution of Sequence Identiy between the two BlastP runs

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.

Figure ??
Distribution of Sequence Id between Psi-Blast runs with 2 iterations vs 10 iterations (using E-Value 10e-10)
Figure ??
Distribution of Sequence Id for Psi-Blast runs with 2 iterations with different E-Values (def E-Value vs E-Value of10e-10)
Figure ??
Distribution of Sequence Id for Psi-Blast runs with 10 iterations with different E-Values (def E-Value vs E-Value of10e-10)
Figure ??
Distribution of logarithmic E_Values for the four different PSIBlast runs

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 ??).

Figure ??
Sequence identity distributions of HHBlits run with 2 and with 8 iterations.
Figure ??
logarithmic E_Value distributions of HHBlits run with 2 and with 8 iterations.

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).

Figure ??
Distribution of overlapping Hits for the eight different used Sequence Searches.


Default E-Values: as could be expected, the normal BLAST search is mostly contained in the PsiBLAST search with two iterations. HHBlits found a large number of different hits, with only 48 out of 274 common hits in common with the BLAST searches.
Taking PsiBLAST with 10 iterations into account brings in a large number of common sequences among the three searches (110), which could be interesting since there seems to be high conversation among them.
Strict E-Values for PsiBLAST and default E-Value for HHBlits with 2 iterations: The number of common hits among all three is now substantially lower, while PsiBLAST with two and ten iterations share a great number of their hits.
Increasing the number of HHBlits-iteration yields more hits for HHBlits, but does not increase the number of common hits with PSI-Blast in 2 or 10 iterations. However, 10 sequences are common and could be interesting for further investigation.

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

# 60-99% sequence identity
Q8BZC2	1.7e-25	90
E1BVP5	e-140	72
H2RVG4	e-141	63
G3VM93	e-105	72
F6ZFQ0	e-139	78
F8WFU8	e-145	86
Q28C61	e-132	68
H2M5L4	e-133	64

# 40-59% sequence identity
G5BTW1	e-133	43
G6FRX8	e-103	39
F7NV91	e-112	39
G1Q6P7	e-120	42
H0WH68	e-135	44
F2PFG6	e-119	40
H2MX25	5e-81	40
Q1Z2X2	e-115	38

# 20-39% sequence identity
Q2F9Q7	e-109	31
Q8YQC1	e-117	41
E1SMZ8	e-108	39
D7E1T3	e-110	36
A5GQV1	7e-92	33
E8LP14	e-107	31
F9TUZ3	e-106	30
A6VUE4	e-101	35


General Results

All in all the three Alignment methods yield comparable results. One can identify several conserved regions. Especially the two groups with sequence identities <60% show very similar MSAs.

There are three strongly conserved motivs located in the first half of the sequences:

  • GGTHGNE
  • DLNR
  • DLHNT

For the second half of the sequence alignments there is no clear concensus about reserved motifs, but several residues are strongly conserved and may be of functional or structural importance.

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.

ClustalW

command: clustalw -align -infile=./db_over60.fa -outfile=./clustalw_msa_60.aln


Jalview Representation of the ClustalW Alignment with the dataset with 20-39% sequence identity. Colored are conserved residues (>65%).
Jalview Representation of the ClustalW Alignment with the dataset with 40-59% sequence identity. Colored are conserved residues (>65%).
Jalview Representation of the ClustalW Alignment with the dataset with 60-100% sequence identity.Colored are conserved residues (>65%).

TCoffee

Jalview Representation of the T-Coffee Alignment with the dataset with 20-39% sequence identity. Colored are conserved residues (>65%).
Jalview Representation of the T-Coffee Alignment with the dataset with 40-59% sequence identity. Colored are conserved residues (>65%).
Jalview Representation of the T-Coffee Alignment with the dataset with 60-100% sequence identity.Colored are conserved residues (>65%).

Muscle

Jalview Representation of the Muscle Alignment with the dataset with 20-39% sequence identity. Colored are conserved residues (>65%).
Jalview Representation of the Muscle Alignment with the dataset with 40-59% sequence identity. Colored are conserved residues (>65%).
Jalview Representation of the Muscle Alignment with the dataset with 60-100% sequence identity.Colored are conserved residues (>65%).

Concerning the wildtype human Aspartoacylase

The three identified motifs can also be foung in the wildtype protein (compare coloring of sequence on top of page). We colored the respective residues in the structure. They all are positioned in the same region of the protein and thus might implicate an important functional region.

Human Aspartoacylse (PDB 2O53) with the motif residues DLHNT
Human Aspartoacylse (PDB 2O53) with the motif residues DLNR
Human Aspartoacylse (PDB 2O53) with the motif residues GGLNR.