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

Overlap of the results of the different sequence searches. (At most 5 different lists could be captured in one diagram).

The overlaps between the results of the different tools (FASTA, BLAST, 4 PSI-BLAST runs) have been investigated and are visualized using Venn-Diagrams (created with <ref>http://bioinformatics.psb.ugent.be/webtools/Venn/</ref>). The results of HHSearch are not included as a different database (pdb70) was used and therefore the results can not be compared.
In total, the 6 different sequence searches returned 626 unique sequences whereof 405 sequences were returned by each of the searches and 41 were only found by a single one. There were no sequences which have only been found by PSI-BLAST runs with 3 iterations whereas FASTA returned 25 sequences that have not been found by any other tool. These numbers indicate that the different sequence searches return very similar sequences and that each of the tools could be used to retrieve the related sequences of glucocerebrosidase.

Sequence Identity

As BLAST only shows details for the 250 "best" alignments, the sequence identitiy could only be analyzed for the 250 first sequences of the BLAST and PSI-BLAST results.

Identity distribution of the different sequence searches.

BLAST

Identity	BLAST	PSI-BLAST 3 iterations e-value cutoff 0.005	PSI-BLAST 3 iterations e-value cutoff 10e-6	PSI-BLAST 5 iterations e-value cutoff 0.005	PSI-BLAST 5 iterations e-value cutoff 10e-6	FASTA	HHSearch
0-9 %	0	0	0	0	0	0	8
10-19%	0	0	0	0	0	0	89
20-29%	43	85	85	96	94	240	2
30-39 %	127	98	98	90	91	160	0
40-49 %	28	26	26	24	25	44	0
50-59 %	6	4	4	3	3	6	0
60-69 %	3	0	0	0	0	3	0
70-79 %	1	1	1	1	1	6	0
80-89 %	15	9	9	9	9	27	0
90-100 %	27	27	27	27	27	34	1
Total	250	250	250	250	250	520	100

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

Tools used

• Cobalt

Cobalt was not yet installed, so we downloaded it here: [[1]]

command
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

command
time clustalw

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

Multiple Alignment by ClustalW in Jalview

• Muscle

command
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

command
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

command
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

Results

Gaps in the reference structure

	Cobalt	ClustalW	Muscle	T-Coffee	3D-Coffee/Expresso
# Gaps	413	404	442	441	546

Conserved Columns

	Cobalt	ClustalW	Muscle	T-Coffee	3D-Coffee/Expresso
>50%	133	133	131	125	123
>60%	98	98	96	104	90
>70%	64	64	62	67	58
>80%	53	54	54	50	57
>90%	52	51	51	52	49
100%	25	23	26	26	25

References

<references />