Glucocerebrosidase sequence alignments

Sequence searches

Several different tools were used in order to look for sequences that are related to glucocerebrosidase in the non-redundant sequence database.

• FASTA

As Fasta was not initially installed, it was downloaded from the EBI FTP Download Site <ref>ftp://ftp.ebi.ac.uk/pub/software/unix/fasta/fasta36/</ref>.

Command:

../bin/fasta36 gbaseq.fasta /data/blast/nr/nr > fasta_gba_search.out

• BLAST

Command:

blastall -p blastp -d /data/blast/nr/nr -i gbaseq.fasta -o blast.out

• PSI-BLAST

This tool was used 4 times with all different combinations of 3 or 5 iterations (x) and an E-value cut-off (y) of 0.005 or 10e-6.

Command:

blastpgp -d /data/blast/nr/nr -i gbaseq.fasta -o psi_blast_x_y.out -j x -h y

Furthermore the online version of HHSearch <ref>http://toolkit.lmb.uni-muenchen.de/hhpred</ref> was used to search against the pdb70 database of May 14th.

Results

The sequence search with FASTA returned 520 sequences. BLAST, as well as the different PSI-BLAST runs returned 500 sequences. The search with HHSearch against the pdb database only resulted in 100 sequences.

Overlap

The overlaps between the results of the different tools applied to the non-redundant sequence database are visualized using Venn-Diagrams<ref>http://bioinformatics.psb.ugent.be/webtools/Venn/</ref>. The results of HHSearch are not included as a different database (pdb70) was used.

Fasta

As Fasta was not initially installed, it was downloaded from the EBI FTP Download Site <ref>ftp://ftp.ebi.ac.uk/pub/software/unix/fasta/fasta36/</ref>.

Command:
../bin/fasta36 gbaseq.fasta /data/blast/nr/nr > fasta_gba_search.out

To apply the tool, the following command was used:

../bin/fasta36 gbaseq.fasta /data/blast/nr/nr > fasta_gba_search.out

Fasta

Fasta was not yet installed, so we downloaded it here: [[1]]
../bin/fasta36 gbaseq.fasta /data/blast/nr/nr > fasta_gba_search.out

identity	percentage	number
0-9 %	0.0	0
10-19%	0.0	0
20-29%	0.462	240
30-39%	0.308	160
40-49%	0.085	44
50-59%	0.012	6
60-69%	0.006	3
70-79%	0.012	6
80-89%	0.052	27
90-100%	0.065	34
Total: 520

Blast

blastall -p blastp -d /data/blast/nr/nr -i gbaseq.fasta -o blast.out

identity	percentage	number
>= 0%	0.0	0
>= 10%	0.0	0
>= 20%	0.172	43
>= 30%	0.508	127
>= 40%	0.112	28
>= 50%	0.024	6
>= 60%	0.012	3
>= 70%	0.004	1
>= 80%	0.06	15
>= 90%	0.108	27
Total: 250

PSI-Blast

3 iterations, E-value cutoff 0.005

blastpgp -d /data/blast/nr/nr -i gbaseq.fasta -o psi_blast_3_0.005.out -j 3 -h 0.005

identity	percentage	number
>= 0%	0.0	0
>= 10%	0.0	0
>= 20%	0.34	85
>= 30%	0.392	98
>= 40%	0.104	26
>= 50%	0.016	4
>= 60%	0.0	0
>= 70%	0.004	1
>= 80%	0.036	9
>= 90%	0.108	27
Total: 250

3 iterations, E-value cutoff 10E-6

blastpgp -d /data/blast/nr/nr -i gbaseq.fasta -o psi_blast_3_10E-6.out -j 3 -h 10E-6

identity	percentage	number
>= 0%	0.0	0
>= 10%	0.0	0
>= 20%	0.34	85
>= 30%	0.392	98
>= 40%	0.104	26
>= 50%	0.016	4
>= 60%	0.0	0
>= 70%	0.004	1
>= 80%	0.036	9
>= 90%	0.108	27
Total: 250

5 iterations, E-value cutoff 0.005

blastpgp -d /data/blast/nr/nr -i gbaseq.fasta -o psi_blast_5_0.005.out -j 5 -h 0.005

identity	percentage	number
>= 0%	0.0	0
>= 10%	0.0	0
>= 20%	0.384	96
>= 30%	0.36	90
>= 40%	0.096	24
>= 50%	0.012	3
>= 60%	0.0	0
>= 70%	0.004	1
>= 80%	0.036	9
>= 90%	0.108	27
Total: 250

5 iterations, E-value cutoff 10E-6

blastpgp -d /data/blast/nr/nr -i gbaseq.fasta -o psi_blast_5_10E-6.out -j 5 -h 10E-6

identity	percentage	number
>= 0%	0.0	0
>= 10%	0.0	0
>= 20%	0.376	94
>= 30%	0.364	91
>= 40%	0.1	25
>= 50%	0.012	3
>= 60%	0.0	0
>= 70%	0.004	1
>= 80%	0.036	9
>= 90%	0.108	27
Total: 250

HHSearch

For HHSearch we used [[2]] with the pdb70 database of May 14th.

Multiple sequence alignments

Sequences used for multiple sequence alignments

For the multiple sequence alignments we used our reference sequence and twenty sequences we had found with sequence searches. We tried to avoid hypothetical sequences and tried to take sequences, that have similiar identities in all sequence searches. The following tables show the chosen sequences with their identities in the different searches. We only found one pdb structure (which was also in the HSSP database).

our reference sequence: P04062, GLCM_HUMAN Glucosylceramidase

99 - 90% sequence identity

NP_001127488.1	glucosylceramidase precursor	Pongo abelii	95.0, 98.0, 98.0, 98.0, 98.0, 98.1
3KE0	A Chain A, Crystal Structure Of N370s Glucocerebrosidase At Acidic Ph.		97.0, 99.0, 99.0, 99.0, 99.0, 99.8
EAW53100.1	glucosidase, beta; acid (includes glucosylceramidase), isoform CRA_a	Homo sapiens	97.0, 99.0, 99.0, 99.0, 99.0, 99.6
NP_001165283.1	glucosylceramidase isoform 3 precursor	Homo sapiens	88.0, 90.0, 90.0, 90.0, 90.0, 90.9
NP_001128784.1	DKFZP469B0323 protein	Pongo abelii	95.0, 97.0, 97.0, 97.0, 97.0, 97.4

89 - 60% sequence identity

NP_032120.1	glucosylceramidase isoform 1	Mus musculus	84.0, 86.0, 86.0, 86.0, 86.0, 86.4
EDL15229.1	glucosidase, beta, acid, isoform CRA_a	Mus musculus	84.0, 86.0, 86.0, 86.0, 86.0, 86.3
NP_001121111.1	glucosidase, beta, acid	Rattus norvegicus	85.0, 87.0, 87.0, 87.0, 87.0, 87.6
NP_001039886.1	glucosylceramidase precursor	Bos taurus	86.0, 89.0, 89.0, 89.0, 89.0, 89.2
NP_001005730.1	glucosylceramidase precursor	Sus scrofa	87.0, 89.0, 89.0, 89.0, 89.0, 89.6

59 - 40% sequence identity

EFN73638.1	Glucosylceramidase	Camponotus floridanus	41.0, 40.0, 40.0, 41.0, 40.0, 42.2
CAG11843.1	unnamed protein product	Tetraodon nigroviridis	52.0, 53.0, 53.0, 53.0, 53.0, 54.2
NP_500785.1	hypothetical protein Y4C6B.6	Caenorhabditis elegans	41.0, 40.0, 39.0, 40.0, 39.0, 41.9
EFA07058.1	hypothetical protein TcasGA2_TC010035	Tribolium castaneum	41.0, 42.0, 41.0, 42.0, 41.0, 43.2
EFO26573.1	O-glycosyl hydrolase family 30 protein	Loa loa	40.0, 40.0, 40.0, 40.0, 40.0, 41.7

39 - 20% sequence identity

ZP_07040024.1	glucosylceramidase	Bacteroides sp. 3_1_23	26.0, 24.0, 24.0, 24.0, 24.0, 25.5
YP_244236.1	glycosyl hydrolase	Xanthomonas campestris pv. campestris str. 8004	33.0, 31.0, 30.0, 31.0, 31.0, 33.4
ZP_01885435.1	glycosyl hydrolase	Pedobacter sp. BAL39	36.0, 33.0, 32.0, 33.0, 33.0, 37.2
ZP_07388379.1	Glucan endo-1,6-beta-glucosidase	Paenibacillus curdlanolyticus YK9	28.0, 24.0, 23.0, 24.0, 24.0, 30.1
NP_623885.1	O-glycosyl hydrolase family protein	Thermoanaerobacter tengcongensis MB4	37.0, 34.0, 33.0, 34.0, 32.0, 37.5

Cobalt

Cobalt was not yet installed, so we downloaded it here: [[3]]

time /home/student/Desktop/ncbi-cobalt-2.0.1/cobalt -i multiple_alignment.fasta -norps T > cobalt_multiple_alignment.aln

	time
real	0m3.488s
user	0m2.320s
sys	0m0.180s

Multiple Alignment by Cobalt in Jalview

ClustalW

time clustalw

	time
real	0m40.625s
user	0m5.320s
sys	0m0.070s

Multiple Alignment by ClustalW in Jalview

Muscle

time muscle -in multiple_alignment.fasta -out muscle_multiple_alignment.aln

	time
real	0m3.018s
user	0m1.710s
sys	0m0.100s

Multiple Alignment by Muscle with Jalview

T-Coffee

time t_coffee multiple_alignment.fasta

	time
real	0m41.360s
user	0m34.000s
sys	0m0.920s

Multiple Alignment by T-Coffee with Jalview

3D-Coffee/Expresso

time t_coffee -seq multiple_alignment.fasta -mode expresso -pdb_type dn

	time
real	12m19.825s
user	5m17.140s
sys	0m46.970s

Multiple Alignment by 3D-Coffee/Expresso in Jalview

References

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