Difference between revisions of "Sequence Alignment GLA"

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<sup>by [[User:Drexler|Benjamin Drexler]] and [[User:Grandke|Fabian Grandke]]</sup>
 
=Sequence Searches=
 
=Sequence Searches=
 
[[alpha_galactosidase_reference_amino_acid|GLA sequence]] was searched in the PDB non-redundant(nr) database using three different tools, that are listed below.
 
[[alpha_galactosidase_reference_amino_acid|GLA sequence]] was searched in the PDB non-redundant(nr) database using three different tools, that are listed below.
Line 53: Line 54:
 
| [[File:GLA_Eval.png|thumb|Figure 4: Distribution of the e-values.]]
 
| [[File:GLA_Eval.png|thumb|Figure 4: Distribution of the e-values.]]
 
| Figure 4 shows the e-value distributions of the programs.
 
| Figure 4 shows the e-value distributions of the programs.
Since all blast based programs had a huge number of e-values of the value 0, it was impossible to plot the logarithm of the distribution correctly.
+
Since all blast based programs had serveral hits with an e-values of 0, it was not possible to plot the logarithm of the distribution correctly. The certain values have been set to -500 to provide a plot at all.
  +
We used the logarithm function to provide a clearlier representation of the e-values. A plot of the original values is allocated on [[Media:GLA_Eval_ugly.png|this]] page.
The certain values have been set to -500 to provide any plot at all.
 
The logarithm function was necessary, because some outliers were so widely spread, that there was no visible distribution([[Media:GLA_Eval_ugly.png|Plot]]).
 
 
As seen before, the results of the PSI-Blast runs are almost equal, but the ones with five iterations produce slightly better e-values.
 
As seen before, the results of the PSI-Blast runs are almost equal, but the ones with five iterations produce slightly better e-values.
 
The Blast results are a little worse and the Fasta runs e-values are even worse.
 
The Blast results are a little worse and the Fasta runs e-values are even worse.
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{| class="centered"; style="width:65%;"
 
{| class="centered"; style="width:65%;"
 
| [[File:GLA_Ident.png|thumb|right|Figure 5: Distribution of the identities.]]
 
| [[File:GLA_Ident.png|thumb|right|Figure 5: Distribution of the identities.]]
| Figure 5 shows the identity distributions of the programs. As not all programs delivered the same number of sequences, the values are normalized to 100. Despite some differences, the majority distribution of the identities is similar, except for HHsearch. The other ones does all have peaks between 30-45% identity.
+
| Figure 5 shows the identity distributions of the programs. As not all programs delivered the same number of sequences, the values are normalized to 100 hits. Despite some differences, the majority distribution of the identities is similar, except for HHsearch. All the other ones have peaks between 30-45% identity.
 
Especially the PSI-Blast results are almost equal. The peaks of Blast and Fasta have a huge overlap, as well.
 
Especially the PSI-Blast results are almost equal. The peaks of Blast and Fasta have a huge overlap, as well.
 
Only the results of HHsearch are outliers to the other distributions.
 
Only the results of HHsearch are outliers to the other distributions.
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|}
 
|}
   
====T-Coffee 3D====
+
===T-Coffee 3D===
 
To start the 3D mode the additional parameters <code>-mode expresso -pdb_type dn</code> were given as a suffix to the command.
 
To start the 3D mode the additional parameters <code>-mode expresso -pdb_type dn</code> were given as a suffix to the command.
   
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| 6
 
| 6
 
|-
 
|-
| Disulfide bond
+
| Disulfide bond<sup>1</sup>
 
| 52 / 94
 
| 52 / 94
 
| 3 / 11
 
| 3 / 11
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| 3 / 11
 
| 3 / 11
 
|-
 
|-
| Disulfide bond
+
| Disulfide bond<sup>1</sup>
 
| 56 / 63
 
| 56 / 63
 
| 0 / 0
 
| 0 / 0
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| 0 / 0
 
| 0 / 0
 
|-
 
|-
| Disulfide bond
+
| Disulfide bond<sup>1</sup>
 
| 142 / 172
 
| 142 / 172
 
| 8 / 11
 
| 8 / 11
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| 7 / 11
 
| 7 / 11
 
|-
 
|-
| Disulfide bond
+
| Disulfide bond<sup>1</sup>
 
| 202 / 223
 
| 202 / 223
 
| 11 / 7
 
| 11 / 7
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| 8 / 7
 
| 8 / 7
 
|-
 
|-
| Disulfide bond
+
| Disulfide bond<sup>1</sup>
 
| 378 / 382
 
| 378 / 382
 
| 4 / 0
 
| 4 / 0
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|-
 
|-
 
|}
 
|}
Since two residues are part of a disulfide bond, the two values are separated by the "/".
+
<sup>1</sup> Since two residues are part of a disulfide bond, the two positions or values are separated by "/".
   
 
First of all, we have a great variety in these results. The residue 231 that is part of the active site has a conservation index of 11 in the MSA of all programs, which is clearly a strong sign of conservation. In contrast, some of the residues which are glycosylated (e.g. residue 139) or establish a disulfide bond (e.g. residues 56 / 63) have conservation indices that are close to 0.
 
First of all, we have a great variety in these results. The residue 231 that is part of the active site has a conservation index of 11 in the MSA of all programs, which is clearly a strong sign of conservation. In contrast, some of the residues which are glycosylated (e.g. residue 139) or establish a disulfide bond (e.g. residues 56 / 63) have conservation indices that are close to 0.
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! T-Coffee
 
! T-Coffee
 
! T-Coffee 3D
 
! T-Coffee 3D
  +
|- style="background: lightgray;"
|-
 
 
|Beta strand || 42 – 46 || 0 || 0 || 0 || 0 || 0
 
|Beta strand || 42 – 46 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Helix || 47 – 50 || 0 || 0 || 0 || 0 || 0
 
|Helix || 47 – 50 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Turn || 56 – 58 || 0 || 0 || 0 || 0 || 0
 
|Turn || 56 – 58 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Turn || 60 – 62 || 0 || 0 || 0 || 0 || 0
 
|Turn || 60 – 62 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgreen;"
|-
 
|Helix || 66 – 78 || 0 || 0 || 0 || 0 || 1
+
|Helix || 66 – 78 || 0 || 0 || 0 || 0 || '''1'''
  +
|- style="background: lightgray;"
|-
 
 
|Helix || 81 – 84 || 0 || 0 || 0 || 0 || 0
 
|Helix || 81 – 84 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Beta strand || 88 – 90 || 0 || 0 || 0 || 0 || 0
 
|Beta strand || 88 – 90 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Turn || 110 – 112 || 0 || 0 || 0 || 0 || 0
 
|Turn || 110 – 112 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Helix || 116 – 126 || 0 || 0 || 0 || 0 || 0
 
|Helix || 116 – 126 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 130 – 140 || 0 || 1 || 0 || 0 || 2
+
|Beta strand || 130 – 140 || 0 || '''1''' || 0 || 0 || '''2'''
  +
|- style="background: lightgray;"
|-
 
 
|Beta strand || 144 – 146 || 0 || 0 || 0 || 0 || 0
 
|Beta strand || 144 – 146 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgreen;"
|-
 
|Turn || 149 – 151 || 0 || 0 || 0 || 0 || 3
+
|Turn || 149 – 151 || 0 || 0 || 0 || 0 || '''3'''
  +
|- style="background: lightgray;"
|-
 
 
|Helix || 152 – 162 || 0 || 0 || 0 || 0 || 0
 
|Helix || 152 – 162 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Beta strand || 166 – 170 || 0 || 0 || 0 || 0 || 0
 
|Beta strand || 166 – 170 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgreen;"
|-
 
|Helix || 177 – 194 || 38 || 0 || 23 || 0 || 34
+
|Helix || 177 – 194 || '''38''' || 0 || '''23''' || 0 || '''34'''
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 199 – 202 || 1 || 1 || 1 || 1 || 0
+
|Beta strand || 199 – 202 || '''1''' || '''1''' || '''1''' || '''1''' || 0
  +
|- style="background: lightgreen;"
|-
 
|Helix || 204 – 208 || 3 || 0 || 3 || 0 || 0
+
|Helix || 204 – 208 || '''3''' || 0 || '''3''' || 0 || 0
  +
|- style="background: lightgreen;"
|-
 
|Turn || 209 – 211 || 0 || 0 || 0 || 0 || 4
+
|Turn || 209 – 211 || 0 || 0 || 0 || 0 || '''4'''
  +
|- style="background: lightgray;"
|-
 
 
|Helix || 216 – 219 || 0 || 0 || 0 || 0 || 0
 
|Helix || 216 – 219 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Turn || 220 – 222 || 0 || 0 || 0 || 0 || 0
 
|Turn || 220 – 222 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Beta strand || 224 – 227 || 0 || 0 || 0 || 0 || 0
 
|Beta strand || 224 – 227 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgreen;"
|-
 
|Helix || 236 – 247 || 0 || 18 || 29 || 22 || 36
+
|Helix || 236 – 247 || 0 || '''18''' || '''29''' || '''22''' || '''36'''
  +
|- style="background: lightgray;"
|-
 
 
|Turn || 248 – 252 || 0 || 0 || 0 || 0 || 0
 
|Turn || 248 – 252 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Helix || 253 – 256 || 0 || 0 || 0 || 0 || 0
 
|Helix || 253 – 256 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 261 – 264 || 0 || 0 || 0 || 0 || 1
+
|Beta strand || 261 – 264 || 0 || 0 || 0 || 0 || '''1'''
  +
|- style="background: lightgray;"
|-
 
 
|Beta strand || 272 – 274 || 0 || 0 || 0 || 0 || 0
 
|Beta strand || 272 – 274 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Helix || 277 – 289 || 0 || 0 || 0 || 0 || 0
 
|Helix || 277 – 289 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Beta strand || 294 – 296 || 0 || 0 || 0 || 0 || 0
 
|Beta strand || 294 – 296 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Helix || 305 – 312 || 0 || 0 || 0 || 0 || 0
 
|Helix || 305 – 312 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Helix || 314 – 320 || 0 || 0 || 0 || 0 || 0
 
|Helix || 314 – 320 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgray;"
|-
 
 
|Beta strand || 329 – 332 || 0 || 0 || 0 || 0 || 0
 
|Beta strand || 329 – 332 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 334 – 343 || 11 || 0 || 0 || 0 || 2
+
|Beta strand || 334 – 343 || '''11''' || 0 || 0 || 0 || '''2'''
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 345 – 355 || 0 || 0 || 0 || 0 || 1
+
|Beta strand || 345 – 355 || 0 || 0 || 0 || 0 || '''1'''
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 359 – 361 || 0 || 0 || 0 || 0 || 1
+
|Beta strand || 359 – 361 || 0 || 0 || 0 || 0 || '''1'''
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 363 – 369 || 20 || 0 || 18 || 58 || 2
+
|Beta strand || 363 – 369 || '''20''' || 0 || '''18''' || '''58''' || '''2'''
  +
|- style="background: lightgreen;"
|-
 
|Turn || 370 – 372 || 0 || 0 || 0 || 0 || 3
+
|Turn || 370 – 372 || 0 || 0 || 0 || 0 || '''3'''
  +
|- style="background: lightgray;"
|-
 
 
|Turn || 374 – 376 || 0 || 0 || 0 || 0 || 0
 
|Turn || 374 – 376 || 0 || 0 || 0 || 0 || 0
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 379 – 383 || 15 || 0 || 5 || 0 || 6
+
|Beta strand || 379 – 383 || '''15''' || 0 || '''5''' || 0 || '''6'''
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 385 – 390 || 0 || 0 || 0 || 0 || 49
+
|Beta strand || 385 – 390 || 0 || 0 || 0 || 0 || '''49'''
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 396 – 400 || 0 || 0 || 0 || 2 || 1
+
|Beta strand || 396 – 400 || 0 || 0 || 0 || '''2''' || '''1'''
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 402 – 407 || 0 || 0 || 0 || 0 || 1
+
|Beta strand || 402 – 407 || 0 || 0 || 0 || 0 || '''1'''
  +
|- style="background: lightgreen;"
|-
 
|Beta strand || 412 – 419 || 0 || 0 || 1 || 1 || 14
+
|Beta strand || 412 – 419 || 0 || 0 || '''1''' || '''1''' || '''14'''
  +
|- style="background: lightblue;"
|-
 
|'''Overall''' || '''-''' || '''88''' || '''20''' || '''80''' || '''84''' || '''161'''
+
|'''Overall number of gaps in a secondary structure''' || '''-''' || '''88''' || '''20''' || '''80''' || '''84''' || '''161'''
  +
|- style="background: lightblue;"
  +
|'''Overall number of gaps''' || '''-''' || '''188''' || '''165''' || '''191''' || '''190''' || '''287'''
  +
|- style="background: lightblue;"
  +
|'''Percentage of gaps<sup>1</sup>''' || '''-''' || '''46.8%''' || '''10.5%''' || '''41.9%''' || '''44.2%''' || '''56.1%'''
 
|-
 
|-
 
|}
 
|}
  +
<sup>1</sup> Percentage of gaps describes the ratio of number of gaps in a secondary structure to the overall number of gaps, e.g. (88 / 188 = 0.468 = 46.8% for Cobalt)
not finished yet...
 
  +
  +
We used the secondary structure annotation of [http://www.uniprot.org/uniprot/P06280 UniProt] to determine the number of gaps in a secondary structure of our reference sequence. It is remarkable that ClustalW has a significant lower number of percentage of gaps. Hence we assume that ClustalW is better able to take secondary structure into consideration than the other programs.
   
 
== References ==
 
== References ==
 
<references />
 
<references />
  +
  +
[[Category:Fabry Disease]]

Latest revision as of 15:11, 30 May 2011

by Benjamin Drexler and Fabian Grandke

Sequence Searches

GLA sequence was searched in the PDB non-redundant(nr) database using three different tools, that are listed below. An additional search has been made applying HHsearch on the pdb70 database(max.70% sequence identity).

Blast

We used the NCBI Blast Version 2.2.18 with the command:

blast -i sequence.fasta -d database -p blastp

Fasta

As no fasta program was installed, we downloaded fasta36 and installed it to the virtual machine. The command to run the program is:

fasta36 sequence.fasta database

PSI-Blast

We used the PSI-Blast version 2.2.18 with the command:

blastpgp -i sequence.fasta -d database -j iterations -h e-value

Parameter

We used the following combinations of parameter to run the program:

  • 3 iterations and e-value threshold of 0.005
  • 3 iterations and e-value threshold of 0.002
  • 3 iterations and e-value threshold of 10e-6
  • 5 iterations and e-value threshold of 0.005
  • 5 iterations and e-value threshold of 0.002
  • 5 iterations and e-value threshold of 10e-6

HHsearch

We used the online tool from Gene Center of the LMU Munich with the default parameters:

  • Database = PDB70
  • Max. number of PSI-BLAST iterations = 3
  • Alignment mode = local

The results are still available: Results.

Overlap

Figure 1 shows the overlap between the results of Blast, Fasta, and PSI-Blast with five iterations and e-values of 10e-6 and 0.005. The most significant values in this diagram are the overlap between all four groups, the overlap between the both PSI-Blast groups and the number of Fasta-only sequences. Figure 2 shows the overlap between the results of PSI-Blast with three/five iterations and e-values of 10e-6 and 0.005. Nearly all sequences have been found by all four groups, so the variation of iteration numbers has not much impact in case of our dataset. Figure 3 shows the overlap between the results of PSI-Blast with five iterations and e-values of 10e-6, 0.002 and 0.005. Variation of e-value threshold has obviously not much influence on the results, the number of sequences that do not appear in different groups is very low. The figures were created using an online resource.

Figure 1: Overlap between Blast-, Fasta- and PSI-Blast results.
Figure 2: Overlap between PSI-Blast results with different iteration numbers.
Figure 3: Overlap between PSI-Blast results with different e-values.

E-value Distribution

Figure 4: Distribution of the e-values.
Figure 4 shows the e-value distributions of the programs.

Since all blast based programs had serveral hits with an e-values of 0, it was not possible to plot the logarithm of the distribution correctly. The certain values have been set to -500 to provide a plot at all. We used the logarithm function to provide a clearlier representation of the e-values. A plot of the original values is allocated on this page. As seen before, the results of the PSI-Blast runs are almost equal, but the ones with five iterations produce slightly better e-values. The Blast results are a little worse and the Fasta runs e-values are even worse. As they are on a completely different level, the HHsearch values are not comparable.

Identity Distribution

Figure 5: Distribution of the identities.
Figure 5 shows the identity distributions of the programs. As not all programs delivered the same number of sequences, the values are normalized to 100 hits. Despite some differences, the majority distribution of the identities is similar, except for HHsearch. All the other ones have peaks between 30-45% identity.

Especially the PSI-Blast results are almost equal. The peaks of Blast and Fasta have a huge overlap, as well. Only the results of HHsearch are outliers to the other distributions.

Runtime Analysis

The runtime of each program was measured by using the command time as a prefix in the commandline.

Program Runtime
Blast 2:40 min
Fasta 5:16 min
PSI-Blast: 3 Iterations: E-value cutoff 10e-6 7:50 min
PSI-Blast: 3 Iterations: E-value cutoff 0.002 7:48 min
PSI-Blast: 3 Iterations: E-value cutoff 0.005 7:55 min
PSI-Blast: 5 Iterations: E-value cutoff 10e-6 13:27 min
PSI-Blast: 5 Iterations: E-value cutoff 0.002 13:06 min
PSI-Blast: 5 Iterations: E-value cutoff 0.005 12:49 min

HSSP

We used a HSSP online resource to find proteins related to alpha galactosidase and choose results, that are found in homo sapiens. The resulting files contained the ID's that were found by HSSP, which were used to classify the hits of the searches (Fasta/Blast/PSI-Blast) as true positives (TP). Afterwards we were able to calculate the sensitivity of the search tools in respect to our query.

Program Sensitivity
Blast 20.9%
Fasta 31.6%
PSI-Blast: 3 Iterations: E-value cutoff 10e-6 24.0%
PSI-Blast: 3 Iterations: E-value cutoff 0.002 24.1%
PSI-Blast: 3 Iterations: E-value cutoff 0.005 24.2%
PSI-Blast: 5 Iterations: E-value cutoff 10e-6 24.0%
PSI-Blast: 5 Iterations: E-value cutoff 0.002 24.5%
PSI-Blast: 5 Iterations: E-value cutoff 0.005 24.5%

The sensitivity varies between 20% and 32%. It is remarkable that the different parameters of PSI-Blast do not have any noticeable impact on the result.<br\> Overall these results are not satisfactory, since these sensitivities are very low. We do not think that this is a good benchmark for the search tools and assume that these values are underestimates because of two reasons. First, it could be the case that the result of the HSSP research is not an exhaustive collection of true positives. Second, we only evaluated one query and hence there is a huge bias. Therefore we encourage you to check out some of the results of the other groups:

Hint: To map the IDs from different databases we used the Uniprot ID Mapping.

Multiple Sequence Alignments

Selection of Sequences

We selected twenty sequences of the Fasta/Blast/PSI-Blast search results which fullfilled the following criteria:

  • about 400-450 amino acids long
  • true positive according to the research with HSSP

Unfortunately we were not able to find a sequence of a PDB structure with an identity between 89%-60%. The selected sequences are listed in the following table. We also added our reference sequence to the multiple sequence alignment.

GenBank Identifier Source Description Organism Identity
99%-90% Sequence Identity
295789486 PDB alpha-galactosidase A, chain A Homo sapiens 99%
62896813 GenBank alpha-galactosidase Homo sapiens 99%
269914455 PDB alpha-galactosidase Homo sapiens 99%
297710567 RefSeq alpha-galactosidase A-like Pongo abelii 96%
296235998 RefSeq alpha-galactosidase A Callithrix jacchus 95%
89%-60% Sequence Identity
301788124 RefSeq alpha-galactosidase A-like Ailuropoda melanoleuca 83%
133778924 RefSeq alpha-galactosidase A Mus musculus 78%
114051916 RefSeq alpha-N-acetylgalactosaminidase Bombyx mori 76%
291190554 RefSeq alpha-galactosidase A Salmo salar 67%
148228315 RefSeq alpha-galactosidase Xenopus laevis 65%
59%-40% Sequence Identity
20151048 PDB alpha-N-acetylgalactosaminidase, chain A Gallus gallus 57%
261824882 PDB alpha-N-acetylgalactosaminidase, chain A Homo sapiens 54%
148229665 RefSeq alpha-N-acetylgalactosaminidase Xenopus laevis 47%
92096920 GenBank NAGA protein Bos taurus 46%
260593558 RefSeq alpha-galactosidase Prevotella veroralis F0319 41%
39%-20% Sequence Identity
51701639 Swiss-Prot alpha-galactosidase precursor Lachancea cidri 38%
74626383 Swiss-Prot alpha-galactosidase B precursor Aspergillus niger 35%
299856763 PDB alpha-galactosidase, chain A Saccharomyces cerevisiae 34%
310699603 GenBank alpha-D-galactopyranosidase Fusarium oxysporum 33%
226293587 Swiss-Prot alpha-galactosidase precursor Torulaspora delbrueckii 31%

Programs

Cobalt

We used NCBI Cobalt version 2.0.1 with the command:

cobalt -i sequences.fasta -norps T

Multiple sequence alignment of the 21 sequences by Cobalt in JalView.

ClustalW

We used ClustalW version 1.83 with the command:

clustalw -infile=sequences.fasta

Multiple sequence alignment of the 21 sequences by ClustalW in JalView.

Muscle

We used Muscle version 3.8.31 with the command:

muscle -in sequences.fasta -out muscle_msa.aln

Multiple sequence alignment of the 21 sequences by Muscle in JalView.

T-Coffee

The basic command to start T-Coffee version 8.99 is:

t_coffee sequences.fasta

Multiple sequence alignment of the 21 sequences by T-Coffee in JalView.

T-Coffee 3D

To start the 3D mode the additional parameters -mode expresso -pdb_type dn were given as a suffix to the command.

Multiple sequence alignment of the 21 sequences by T-Coffee 3D in JalView.

Results

Conservation

We used the conservation index according to Livingstone C.D. and Barton G.J.<ref name=livingstone>Livingstone C.D. and Barton G.J. (1993), "Protein Sequence Alignments: A Strategy for the Hierarchical Analysis of Residue Conservation.", CABIOS Vol. 9 No. 6 (745-756)), PubMed</ref> to determine whether a residue is conserved or not. The conservation index was calculated by JalView and ranges from 0 to 11.

Program >= 8 >= 9 >= 10 >= 11
Cobalt 88 70 46 36
ClustalW 87 70 44 36
Muscle 87 69 46 36
T-Coffee 90 72 47 37
T-Coffee 3D 83 60 39 33

Quite obviously the number of conserved residues decreases with an increasing threshold of the conservation index. Overall all programs achieve a similar number of conserved resdiues. Only T-Coffee 3D has lower numbers for all thresholds. This could be due to the fact that T-Coffee 3D tries to incorporate structural information. But the higher the threshold of the conservation index, the smaller the difference. Hence it is very likely that the strongly conserved residues are also existing in the MSA of T-Coffee 3D.

Functionally Important Residues

We used the sequence annotation of UniProt to determine functionally important residues. These residues and their corresponding conservation index in the multiple sequence alignment are listed in the following table. Additionally we also calculated the average conservation index of all positions which are not a gap in the consensus sequence. We can use this value to evaluate whether a functionally important residue has the tendency to be conserved or not.

Type Position Cobalt ClustalW Muscle T-Coffee T-Coffee 3D
Average - 3.44 3.52 3.52 3.53 3.27
Active Site 170 5 5 5 5 5
Active Site 231 11 11 11 11 11
Glycosylation 139 2 2 2 2 2
Glycosylation 192 4 4 4 4 4
Glycosylation 215 0 3 0 3 3
Glycosylation 408 7 7 0 7 6
Disulfide bond1 52 / 94 3 / 11 3 / 11 3 / 11 3 / 11 3 / 11
Disulfide bond1 56 / 63 0 / 0 1 / 0 2 / 2 0 / 0 0 / 0
Disulfide bond1 142 / 172 8 / 11 9 / 11 8 / 11 8 / 11 7 / 11
Disulfide bond1 202 / 223 11 / 7 11 / 7 11 / 7 11 / 7 8 / 7
Disulfide bond1 378 / 382 4 / 0 0 / 0 0 / 0 0 / 0 3 / 1

1 Since two residues are part of a disulfide bond, the two positions or values are separated by "/".

First of all, we have a great variety in these results. The residue 231 that is part of the active site has a conservation index of 11 in the MSA of all programs, which is clearly a strong sign of conservation. In contrast, some of the residues which are glycosylated (e.g. residue 139) or establish a disulfide bond (e.g. residues 56 / 63) have conservation indices that are close to 0. Overall the glycosylated residues seem to have no tendency to be conservated in the MSA, since a vast majority of their values range between 0 and 4. The story is quite different for the residues that establish a disulfide bond. It is remarkable that there is a correlation between the conservation indices of a residue pair, e.g. one residue has a high conservation index when the other residue also has a high conservation index. This obversation makes sense in respect to the biological contact, because the disulfide bond needs both residues to be established. The residues 52 / 94 are the only exception to this.

Number of Gaps

Program Length of MSA Number of gaps (consensus) Number of gaps (reference)
Cobalt 586 69 188
ClustalW 563 46 165
Muscle 589 74 191
T-Coffee 588 64 190
T-Coffee 3D 685 163 287

We determined the number of gaps in the consensus sequence and also in the reference sequence. The length of the MSA and the number of gaps does not differ much among the programs except for T-Coffee 3D. This could be due to the fact that T-Coffee 3D tries to incorporate structural information.

Gaps in Secondary Structure

Secondary Structure Element Positions Cobalt ClustalW Muscle T-Coffee T-Coffee 3D
Beta strand 42 – 46 0 0 0 0 0
Helix 47 – 50 0 0 0 0 0
Turn 56 – 58 0 0 0 0 0
Turn 60 – 62 0 0 0 0 0
Helix 66 – 78 0 0 0 0 1
Helix 81 – 84 0 0 0 0 0
Beta strand 88 – 90 0 0 0 0 0
Turn 110 – 112 0 0 0 0 0
Helix 116 – 126 0 0 0 0 0
Beta strand 130 – 140 0 1 0 0 2
Beta strand 144 – 146 0 0 0 0 0
Turn 149 – 151 0 0 0 0 3
Helix 152 – 162 0 0 0 0 0
Beta strand 166 – 170 0 0 0 0 0
Helix 177 – 194 38 0 23 0 34
Beta strand 199 – 202 1 1 1 1 0
Helix 204 – 208 3 0 3 0 0
Turn 209 – 211 0 0 0 0 4
Helix 216 – 219 0 0 0 0 0
Turn 220 – 222 0 0 0 0 0
Beta strand 224 – 227 0 0 0 0 0
Helix 236 – 247 0 18 29 22 36
Turn 248 – 252 0 0 0 0 0
Helix 253 – 256 0 0 0 0 0
Beta strand 261 – 264 0 0 0 0 1
Beta strand 272 – 274 0 0 0 0 0
Helix 277 – 289 0 0 0 0 0
Beta strand 294 – 296 0 0 0 0 0
Helix 305 – 312 0 0 0 0 0
Helix 314 – 320 0 0 0 0 0
Beta strand 329 – 332 0 0 0 0 0
Beta strand 334 – 343 11 0 0 0 2
Beta strand 345 – 355 0 0 0 0 1
Beta strand 359 – 361 0 0 0 0 1
Beta strand 363 – 369 20 0 18 58 2
Turn 370 – 372 0 0 0 0 3
Turn 374 – 376 0 0 0 0 0
Beta strand 379 – 383 15 0 5 0 6
Beta strand 385 – 390 0 0 0 0 49
Beta strand 396 – 400 0 0 0 2 1
Beta strand 402 – 407 0 0 0 0 1
Beta strand 412 – 419 0 0 1 1 14
Overall number of gaps in a secondary structure - 88 20 80 84 161
Overall number of gaps - 188 165 191 190 287
Percentage of gaps1 - 46.8% 10.5% 41.9% 44.2% 56.1%

1 Percentage of gaps describes the ratio of number of gaps in a secondary structure to the overall number of gaps, e.g. (88 / 188 = 0.468 = 46.8% for Cobalt)

We used the secondary structure annotation of UniProt to determine the number of gaps in a secondary structure of our reference sequence. It is remarkable that ClustalW has a significant lower number of percentage of gaps. Hence we assume that ClustalW is better able to take secondary structure into consideration than the other programs.

References

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