DAP method

BLASTP

• FASTA SEQUENCE FROM NCBI ENTREZ protein = 2IJZ_A

• Origin of query sequence = Pseudomonas aeruginosa

>gi|119390187|pdb|2IJZ|A Chain A, Crystal Structure Of Aminopeptidase
RAELNQGLIDFLKASPTPFHATASLARRLEAAGYRRLDERDAWHTETGGRYYVTRNDSSLIAIRLGRRSP
LESGFRLVGAHTDSPCLRVKPNPEIARNGFLQLGVEVYGGALFAPWFDRDLSLAGRVTFRANGKLESR
LVDFRKAIAVIPNLNIHLNRAANEGWPINAQNELPPIIAQLAPGEAADFRLLLDEQLLREHGITADVVLDYE
LSFYDTQSAAVVGLNDEFIAGARLDNLLSCHAGLEALLNAEGDENCILVCTDHEEVGSCSHCGADGPFLE
QVLRRLLPEGDAFSRAIQRSLLVSADNAHGVHPNYADRHDANHGPALNGGPVIKINSNQRYATNSETA
GFFRHLCQDSEVPVQSFVTRSDMGCGSTIGPITASQVGVRTVDIGLPTFAMHSIRELAGSHDLAHLVKVLGA
FYASSELP

• Performed blastp search against non-redundant (nr) databases which was provided on the CD provided. Query sequences used was Pseudomonas Aeruginosa chain A crystal structure of asparytl aminopeptidase.

• Initial sequence alignment was performed using ClustalX and edited to reduce gapping in the alignment and final multiple sequence alignment was again performed with 38 sequences.

• Treeview32 software was used to view phylogenetic tree produced from multiple sequence alignment and a bootstrapped N-J tree was produced using Clustalx for branches reliability indications.

As mentioned in the methods and website :

C:\blast\blastall -p blastp -d C:\blast\databases\nr -i yourfile.fasta -o usefuloutputname.html

Obtained fastaformat files

C:\blast\fastacmd -d C:\blast\databases\nr -i filewith_img_numbers -o C:\newsequences.fasta

Inputs used for obtaining fastaformat files:

pdb|2IJZ|A 
ref|YP_789908.1|
ref|YP_261475.1|  
ref|ZP_00416764.1|
ref|NP_743887.1|
ref|NP_793647.1|
ref|YP_607123.1|
ref|YP_958321.1|
ref|ZP_01894798.1|
ref|ZP_01166960.1|
ref|ZP_01738318.1|
ref|YP_436072.1| 
ref|ZP_01462550.1|
ref|YP_630602.1|
ref|YP_001615044.1|
ref|YP_747571.1|
ref|YP_113441.1|
ref|XP_001751765.1|
ref|XP_001641062.1|
ref|XP_713998.1|
gb|AAM61631.1|
ref|XP_365906.1|
ref|XP_843934.1|
ref|NP_001045513.1|
ref|XP_001566576.1|
ref|XP_001877081.1| 
gb|ACC64563.1| 
ref|XP_001492028.1|
ref|NP_001039417.1|
ref|YP_833603.1|
ref|NP_036232.2|
ref|NP_001012937.1|
ref|NP_001104301.1|
gb|EDL75426.1|
ref|NP_001085525.1|
ref|XP_462175.1|
ref|NP_956447.1|

Changed headings in every single obtained fasta sequences into organism names only, e.g :

From

>gi|116051260|ref|YP_789908.1| putative aminopeptidase 2 [Pseudomonas aeruginosa UCBPP-PA14]

MRAELNQGLIDFLKASPTPFHATASLARRLEAAGYRRLDERDAWHTEAGGRYYVTRNDSSLIAIRLGRRSPLESGFRLVG

AHTDSPCLRVKPNPEIARNGFLQLGVEVYGGALFAPWFDRDLSLAGRVTFRANGKLESRLVDFRKAIAVIPNLAIHLNRA

ANEGWPINAQNELPPIIAQLAPGEAADFRLLLDEQLLREHGITADVVLDYELSFYDTQSAAVVGLNDEFIAGARLDNLLS

CHAGLEALLNAEGDENCILVCTDHEEVGSCSHCGADGPFLEQVLRRLLPEGDAFSRAIQRSLLVSADNAHGVHPNYADK

DANHGPALNGGPVIKINSNQRYATNSETAGFFRHLCQDSEVPVQSFVTRSDMGCGSTIGPITASQVGVRTVDIGLPTFAM

HSIRELAGSHDLAHLVKVLGAFYASSELP

To

>Pseudomonas_aeruginosa

MRAELNQGLIDFLKASPTPFHATASLARRLEAAGYRRLDERDAWHTEAGGRYYVTRNDSSLIAIRLGRRSPLESGFRLVG

AHTDSPCLRVKPNPEIARNGFLQLGVEVYGGALFAPWFDRDLSLAGRVTFRANGKLESRLVDFRKAIAVIPNLAIHLNRA

ANEGWPINAQNELPPIIAQLAPGEAADFRLLLDEQLLREHGITADVVLDYELSFYDTQSAAVVGLNDEFIAGARLDNLLS

CHAGLEALLNAEGDENCILVCTDHEEVGSCSHCGADGPFLEQVLRRLLPEGDAFSRAIQRSLLVSADNAHGVHPNYADK

DANHGPALNGGPVIKINSNQRYATNSETAGFFRHLCQDSEVPVQSFVTRSDMGCGSTIGPITASQVGVRTVDIGLPTFAM

HSIRELAGSHDLAHLVKVLGAFYASSELP

saved into a new file organismnames.fasta

CLUSTALX

Multiple Sequence Alignment example

Used ClustalX 1.83multiple alignment software tool to align C:\3rdplaceoutnames.fasta. Output format options was changed to NODE before bootstrapping, this is performed in order see reliability of branches in treeview.

Conserved regions (*) of >gi|119390187|pdb|2IJZ|A Chain A, Crystal Structure Of Aminopeptidase was noted for structural analysis.

Output obtained : .aln file (alignment) and .dnd file (output guide tree)

Bootstrapping : .phb file obtained

Treeview

Used Treeview to visualize Phylogenetic tree:

1. Radial Tree
2. Rectangular Cladogram

The results from the blast search were then screened and a selection was of these results were used for a multiple sequence alignment using ClustalX. This result was boostrapped and these values checked and more sequences were added to improve the resolution of specific branches. A bootstrapped phylogram was produced, as well as a radial tree.