Task 2: Multiple Sequence Alignment

Sorry, were behind scedule, page will be filled with content as soon as possible.

We researched the protein sequence of the branched-chain alpha-keto acid dehydrogenase complex subunit alpha (BCKDHA) with the following original sequence:

• BCKDHA

>sp|P12694|ODBA_HUMAN 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial OS=Homo sapiens GN=BCKDHA PE=1 SV=2
MAVAIAAARVWRLNRGLSQAALLLLRQPGARGLARSHPPRQQQQFSSLDDKPQFPGASAE
FIDKLEFIQPNVISGIPIYRVMDRQGQIINPSEDPHLPKEKVLKLYKSMTLLNTMDRILY
ESQRQGRISFYMTNYGEEGTHVGSAAALDNTDLVFGQYREAGVLMYRDYPLELFMAQCYG
NISDLGKGRQMPVHYGCKERHFVTISSPLATQIPQAVGAAYAAKRANANRVVICYFGEGA
ASEGDAHAGFNFAATLECPIIFFCRNNGYAISTPTSEQYRGDGIAARGPGYGIMSIRVDG
NDVFAVYNATKEARRRAVAENQPFLIEAMTYRIGHHSTSDDSSAYRSVDEVNYWDKQDHP
ISRLRHYLLSQGWWDEEQEKAWRKQSRRKVMEAFEQAERKPKPNPNLLFSDVYQEMPAQL
RKQQESLARHLQTYGEHYPLDHFDK

Blast

To calculate the sequence alignments we used the blast and psiblast binaries from NCBI (version 2.2.26+) As the standard blast alignment hits the limit of 250 matches per alignment, and all of them still seemed very significant (Evalue of < 1e-60) we increased the number of max target hits to 2000 and set an Evalue threshold of 0.002. With this method we found about 1550 matching alignments.

As can be seen in the figure to the right

Distibution of sequence similarity with the BCKDHA blast-query against the big80 database.

, the sequence alignments mainly have a similarity between 15% and 40%.

Multiple Sequence Alignment (MSA)

In this task we are to produce MSA´s out of our database search results. The first step here is to create representative datasets, followed by creating MSA´s using different tools, and finally review the alignments using jalview and compare the tools against each other.

Dataset creation

We have chosen the following sequences from the Psi-Blast run with evalue E-10 and 10 iterations, trying to fit into the scheme given on the task-page:

Marinobacter sp. ELB17F2GCR2_ALTMD Alpha keto acid dehydrogenase complex, E1 component, alpha subunit OS=Alteromonas macleodii (strain DSM 17117 / Deep ecotype)

Identifier	Identity	Organism	Description
ref seq
P12694	100%	human	ODBA_HUMAN 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial
high identity ( >60% )
B4DP47	90%	human	B4DP47_HUMAN Uncharacterized protein
H2L9X9	80%	Oryzias latipes	H2L9X9_ORYLA Uncharacterized protein
H2NYX7	90%	Pongo abelii	H2NYX7_PONAB Uncharacterized protein
G3RDZ3	99%	Gorilla gorilla gorilla	G3RDZ3_GORGO Uncharacterized protein
G7PXN6	92%	Macaca fascicularis	G7PXN6_MACFA Putative uncharacterized protein
C1BZX0	61%	Caligus clemensi	C1BZX0_9MAXI 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial
E9C2J8	62%	Capsaspora owczarzaki	E9C2J8_CAPO3 Branched chain keto acid dehydrogenase E1
A8XMR6	63%	Caenorhabditis briggsae	A8XMR6_CAEBR Putative uncharacterized protein
F1L131	61%	Ascaris suum	F1L131_ASCSU 2-oxoisovalerate dehydrogenase subunit alpha
B3S5B4	66%	Trichoplax adhaerens	B3S5B4_TRIAD Putative uncharacterized protein
moderate identity ( >40% )
F7NS20	50%	Rheinheimera sp. A13L	F7NS20_9GAMM Pyruvate/2-oxoglutarate dehydrogenase complex, dehydrogenase component alpha subunit
A3JES2	50%		A3JES2_9ALTE 2-oxoglutarate dehydrogenase complex, dehydrogenase (E1) component, eukaryotic type, alpha subunit
F2GCR2	49%	Alteromonas macleodii (strain DSM 17117 / Deep ecotype)
E1VBY4	48%	Halomonas elongata	E1VBY4_HALED 3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring)
A9TD44	47%	Physcomitrella patens subsp. patens	A9TD44_PHYPA Predicted protein
E1ZBL6	51%	Chlorella variabilis	E1ZBL6_CHLVA Putative uncharacterized protein
F2Q107	51%	Trichophyton equinum	F2Q107_TRIEC 2-oxoisovalerate dehydrogenase subunit alpha
B6GYK7	52%	Penicillium chrysogenum	B6GYK7_PENCW Pc12g08790 protein
G2QH91	53%	Thielavia heterothallica	G2QH91_THIHA Dehydrogenase-like protein
G0S8V2	51%	Chaetomium thermophilum	G0S8V2_CHATD Alpha subunit-like protein
whole range identity ( 0-100% )
F6EVQ9	39%	Sphingobium chlorophenolicum	F6EVQ9_SPHCR 3-methyl-2-oxobutanoate dehydrogenase
B1YII6	39%	Exiguobacterium sibiricum	B1YII6_EXIS2 Pyruvate dehydrogenase
Q9HN77	34%	Halobacterium salinarium	Q9HN77_HALSA Pyruvate dehydrogenase
G2QH91	53%	Thielavia heterothallica	G2QH91_THIHA Dehydrogenase-like protein
A9TD44	47%	Physcomitrella patens subsp. patens	A9TD44_PHYPA Predicted protein
G0S8V2	51%	Chaetomium thermophilum	G0S8V2_CHATD Alpha subunit-like protein
B4DP47	90%	human	B4DP47_HUMAN Uncharacterized protein
H2L9X9	80%	Oryzias latipes	H2L9X9_ORYLA Uncharacterized protein
H2NYX7	90%	Pongo abelii	H2NYX7_PONAB Uncharacterized protein
C1BZX0	61%	Caligus clemensi	C1BZX0_9MAXI 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial

Results

Jalview representations of the alignments:

>60% fraction

ClustalW MSA >60%

T-Coffee MSA >60%

Muscle MSA >60%

>40% fraction

ClustalW MSA >40%

T-Coffee MSA >40%

Muscle MSA >40%

whole range fraction

ClustalW MSA 0-100%

T-Coffee MSA 0-100%

Muscle MSA 0-100%

clustalw >60 ID	gaps
tr|C1BZX0|C1BZX0_9MAXI	84
sp|P12694|ODBA_HUMAN	70
tr|B3S5B4|B3S5B4_TRIAD	174
tr|H2L9X9|H2L9X9_ORYLA	66
tr|H2NYX7|H2NYX7_PONAB	52
tr|F1L131|F1L131_ASCSU	74
tr|B4DP47|B4DP47_HUMAN	67
tr|G7PXN6|G7PXN6_MACFA	32
tr|G3RDZ3|G3RDZ3_GORGO	90
tr|E9C2J8|E9C2J8_CAPO3	78
tr|A8XMR6|A8XMR6_CAEBR	83
conserved >8	235
conserved >10	130

ID	gaps
UniProt/Swiss-Prot|P12694|Q16034|Q16472|ODBA_HUMAN/1-445	80
UniProt/Swiss-Prot|C1BZX0|C1BZX0_9MAXI/1-431	94
UniProt/Swiss-Prot|A8XMR6|A8XMR6_CAEBR/1-432	93
UniProt/Swiss-Prot|B4DP47|E7EW46|B4DP47_HUMAN/1-448	77
UniProt/Swiss-Prot|H2L9X9|H2L9X9_ORYLA/1-449	76
UniProt/Swiss-Prot|F1L131|F1L131_ASCSU/1-441	84
UniProt/Swiss-Prot|G3RDZ3|G3RDZ3_GORGO/1-425	100
UniProt/Swiss-Prot|B3S5B4|B3S5B4_TRIAD/1-341	184
UniProt/Swiss-Prot|G7PXN6|G7PXN6_MACFA/1-483	42
UniProt/Swiss-Prot|H2NYX7|H2NYX7_PONAB/1-463	62
UniProt/Swiss-Prot|E9C2J8|E9C2J8_CAPO3/1-437	88
conserved >8	235
conserved >10	129

ID	gaps
UniProt/Swiss-Prot|P12694|Q16034|Q16472|ODBA_HUMAN/1-445	75
UniProt/Swiss-Prot|C1BZX0|C1BZX0_9MAXI/1-431	89
UniProt/Swiss-Prot|A8XMR6|A8XMR6_CAEBR/1-432	88
UniProt/Swiss-Prot|B4DP47|E7EW46|B4DP47_HUMAN/1-448	72
UniProt/Swiss-Prot|H2L9X9|H2L9X9_ORYLA/1-449	71
UniProt/Swiss-Prot|F1L131|F1L131_ASCSU/1-441	79
UniProt/Swiss-Prot|G3RDZ3|G3RDZ3_GORGO/1-425	95
UniProt/Swiss-Prot|B3S5B4|B3S5B4_TRIAD/1-341	179
UniProt/Swiss-Prot|G7PXN6|G7PXN6_MACFA/1-483	37
UniProt/Swiss-Prot|H2NYX7|H2NYX7_PONAB/1-463	57
UniProt/Swiss-Prot|E9C2J8|E9C2J8_CAPO3/1-437	83
conserved >8	236
conserved >10	130