HANDSON

1
HANDS-ON
2
Presenting The Protein
CADHF_XENLA

• A Member of the cadherin superfamily, which is
  very
• large and includes all presneting at least 1
  cadherin
• domain (usually more)
• Protein includes 5 cadherin extra-celluar
  domains, a
• transmembrane region and an additioncytoplasmic
  domain

Cadherin domains
3
Presenting The Protein
CADHF_XENLA

• Protein function interaction with cadherins
  from an
• adjacent cell in order to form adhesion
  contacts
• A crystal structure exists for the
  extra-cellular region
• shows the basis of dimerization of cadherins
  from
• adajecnt cells.

4
Outline
1. Pairwise Alignment

• 2. Getting Sequences MSA
• Option 1 Got Sequences Only MSA
• Option 2 BLAST then MSA
• Option 3 ConSeq Does It All

3. Alignment Visualization Handeling
4. Applications
5
Pairwise Alignment
Cadherins extra-cellular regions CADHF_XENLA
CADH1_MOUSE
Sequence 1
ltP33148CADHF_XENLA MGSTRLRNASVWLCGLLCLLQVVPSINADV
SGCKPGFSSAEYIFSVNRRELERGRKLGKVNFSDCTTRKHGLYDVGDSRF
RVLPDGTVLVKRHVKLHKDTKFTISTWDARGIKHSTNIAVASKRHRSGEE
AHSRSSKLPVLTFPETHTGLKRKKRDWVIPPIKVSENERGPFPKRLVQIK
SNKDRFNKVYYSITGQGADNPPQGVFRIEWETGWMLVTRPLDREEYDKYV
LSSHAVSENGSPVEEPMEITINVIDQNDNRPKFTQDVFRGSVREGVQPGT
QVMAVSATDEDDNIDSLNGVLSYSILKQDPEEPIPNLFTINRETGVISLI
GTGLDREKFPEYTLTVQATDLEGAGLSVEGKAIIQITDANDNAPIFDPKT
YTALVPENEIGFEVQRLSVTDLDMPGTPAWQAVYKIRVNEGGFFNITTDP
ESNQGILTTAKGLDFELRKQYVLQITVENAEPFSVPLPTSTATVTVTVED
VNEAPFFVPAVSRVDVSEDLSRGEKIISLVAQDPDKQQIQKLSYFIGNDP
ARWLTVNKDNGIVTGNGNLDRESEYVKNNTYTVIMLVTDDGVSVGTGTGT
LILHVLDVNDNGPVPSPRVFTMCDQNPEPQVLTISDADIPPNTYPYKVSL
SHGSDLTWKAELDSKGTSMLLSPTQQLKKGYSIYVLLSDAQNNPQLTVVN
ATVCSCEGKAIKCQEKLVGGFDL
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Pairwise Alignment
Cadherins extra-cellular regions CADHF_XENLA
CADH1_MOUSE
Sequence 2
ltP09803CADH1_MOUSE MGARCRSFSALLLLLQVSSWLCQELEPESC
SPGFSSEVYTFPVPERHLERGHVLGRVRFEGCTGRPRTAFFSEDSRFKVA
TDGTITVKRHLKLHKLETSFLVRARDSSHRELSTKVTLKSMGHHHHRHHH
RDPASESNPELLMFPSVYPGLRRQKRDWVIPPISCPENEKGEFPKNLVQI
KSNRDKETKVFYSITGQGADKPPVGVFIIERETGWLKVTQPLDREAIAKY
ILYSHAVSSNGEAVEDPMEIVITVTDQNDNRPEFTQEVFEGSVAEGAVPG
TSVMKVSATDADDDVNTYNAAIAYTIVSQDPELPHKNMFTVNRDTGVISV
LTSGLDRESYPTYTLVVQAADLQGEGLSTTAKAVITVKDINDNAPVFNPS
TYQGQVPENEVNARIATLKVTDDDAPNTPAWKAVYTVVNDPDQQFVVVTD
PTTNDGILKTAKGLDFEAKQQYILHVRVENEEPFEGSLVPSTATVTVDVV
DVNEAPIFMPAERRVEVPEDFGVGQEITSYTAREPDTFMDQKITYRIWRD
TANWLEINPETGAIFTRAEMDREDAEHVKNSTYVALIIATDDGSPIATGT
GTLLLVLLDVNDNAPIPEPRNMQFCQRNPQPHIITILDPDLPPNTSPFTA
ELTHGASVNWTIEYNDAAQESLILQPRKDLEIGEYKIHLKLADNQNKDQV
TTLDVHVCDCEGTVNNCMK
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Pairwise Alignment
http//www.ebi.ac.uk/emboss/align/
Type of algorithm
Gap opening
Matrix
Protein/DNA
Sequence 1
Gap extension
Sequence 2
8
Pairwise Alignment
Needlman-Wunch Global Alignment
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Pairwise Alignment
Cadherins extra-cellular regions CADHF_XENLA
CADH1_MOUSE
Sequence 1- 5 domains
ltP33148CADHF_XENLA DWVIPPIKVSENERGPFPKRLVQIKSNKDR
FNKVYYSITGQGADNPPQGVFRIEWETGWMLVTRPLDREEYDKYVLSSHA
VSENGSPVEEPMEITINVIDQNDNRPKFTQDVFRGSVREGVQPGTQVMAV
SATDEDDNIDSLNGVLSYSILKQDPEEPIPNLFTINRETGVISLIGTGLD
REKFPEYTLTVQATDLEGAGLSVEGKAIIQITDANDNAPIFDPKTYTALV
PENEIGFEVQRLSVTDLDMPGTPAWQAVYKIRVNEGGFFNITTDPESNQG
ILTTAKGLDFELRKQYVLQITVENAEPFSVPLPTSTATVTVTVEDVNEAP
FFVPAVSRVDVSEDLSRGEKIISLVAQDPDKQQIQKLSYFIGNDPARWLT
VNKDNGIVTGNGNLDRESEYVKNNTYTVIMLVTDDGVSVGTGTGTLILHV
LDVNDNGPVPSPRVFTMCDQNPEPQVLTISDADIPPNTYPYKVSLSHGSD
LTWKAELDSKGTSMLLSPTQQLKKGYSIYVLLSDAQNNPQLTVVNATVCS
CEGKAIKCQEKLVGGFDL
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Pairwise Alignment
Cadherins extra-cellular regions CADHF_XENLA
CADH1_MOUSE
Sequence 2- 2 of the 5 domains
ltP09803CADH1_MOUSE DWVIPPISCPENEKGEFPKNLVQIKSNRDK
ETKVFYSITGQGADKPPVGVFIIERETGWLKVTQPLDREAIAKYILYSHA
VSSNGEAVEDPMEIVITVTDQNDNRPEFTQEVFEGSVAEGAVPGTSVMKV
SATDADDDVNTYNAAIAYTIVSQDPELPHKNMFTVNRDTGVISVLTSGLD
RESYPTYTLVVQAADLQGEGLSTTAKAVITVKD
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Pairwise Alignment
http//www.ebi.ac.uk/emboss/align/
Type of algorithm
Gap opening
Matrix
Protein/DNA
Sequence 1
Gap extension
Sequence 2
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Pairwise Alignment
Smith-Waterman Local Alignment
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Outline
1. Pairwise Alignment

• 2. Getting Sequences MSA
• Option 1 Got Sequences Only MSA
• Option 2 BLAST then MSA
• Option 3 ConSeq Does It All

3. Alignment Visualization Handeling
4. Applications
14
Getting Sequences MSA
Option 1 Got Sequences Only Align

• Servers are available
• ClustalW
• MUSCLE
• T-coffee
• NOTE FASTA format is always acceptable-

gtmaya ISVERYNICE gtschushan IWVEWYLARGE
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Getting Sequences MSA
Option 1 Got Sequences Only MSA
http//www.ebi.ac.uk/Tools/clustalw2/index.html
Default parameters
Sequences
Run
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Getting Sequences MSA
Option 1 Got Sequences Only MSA
View MSA
MSA file
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Getting Sequences MSA
Option 1 Got Sequences Only MSA
http//www.ebi.ac.uk/muscle/
Sequences
Run
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Getting Sequences MSA
Option 1 Got Sequences Only MSA
http//www.ebi.ac.uk/t-coffee/
Sequences
Run
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Getting Sequences MSA
Option 2 BLAST then MSA

• Leading servers both have severe limitations
• BLAST at ncbi-
• 2.BLAST at EBI

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Getting Sequences MSA
Option 2 BLAST then MSA-NCBI
http//www.ncbi.nlm.nih.gov/blast/Blast.cgi
BLASTp
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Getting Sequences MSA
Option 2 BLAST then MSA-NCBI
http//www.ncbi.nlm.nih.gov/blast/Blast.cgi
Query Sequence
Database
Run
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Getting Sequences MSA
Option 2 BLAST then MSA-NCBI
http//www.ncbi.nlm.nih.gov/blast/Blast.cgi
Mark all
Mark only wanted
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Getting Sequences MSA
Option 2 BLAST then MSA-NCBI
http//www.ncbi.nlm.nih.gov/blast/Blast.cgi
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Getting Sequences MSA
Option 2 BLAST then MSA-NCBI
http//www.ncbi.nlm.nih.gov/blast/Blast.cgi
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Getting Sequences MSA
Option 2 BLAST then MSA-EBI
http//www.ebi.ac.uk/blastall/index.html
Many datbases, including UniProt
Get maximum number of alignments!
Insert sequence
RUN
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Getting Sequences MSA
Option 2 BLAST then MSA-EBI
http//www.ebi.ac.uk/blastall/index.html
Mark all or wanted
Send sequences to ClustalW
Get sequences
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Getting Sequences MSA
Option 2 BLAST then MSA

• Leading servers both have severe limitations
• BLAST at ncbi-
• - Cant BLAST against Uniprot, only SwissProt
• - Only the full sequences can be extracted
• 2.BLAST at EBI
• - BLAST against UniProt
• - Can go straight to MSA
• - Only the full sequences can be extracted
• - The number of presented sequences is limited

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Getting Sequences MSA
Option 3 ConSeq Does It All

• ConSeq calculates evolutionary conservation
  scores
• An MSA is needed
• can be provided or calculated automatically
• Advantages
• BLASTs UniProt (unlike BLAST ncbi)
• No restriction on sequences number (unlike BLAST
  ebi)
• Takes the found fragments (unlike BLAST ncbi
  ebi)

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Getting Sequences MSA
Option 3 ConSeq Does It All
http//conseq.bioinfo.tau.ac.il/
Query sequence
MSA algorithm
BLAST options
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Getting Sequences MSA
Option 3 ConSeq Does It All
http//conseq.bioinfo.tau.ac.il/
Homologues
MSA!
http//conseq.bioinfo.tau.ac.il/results/1199823726
/output.html
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Outline
1. Pairwise Alignment

• 2. Getting Sequences MSA
• Option 1 Got Sequences Only MSA
• Option 2 BLAST then MSA
• Option 3 ConSeq Does It All

3. Alignment Visualization Handeling
4. Applications
32
Alignment Visualization Handeling
Easiest Using Bioedit
http//www.mbio.ncsu.edu/BioEdit/bioedit.html
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Alignment Visualization Handeling
BoxShade
Choose ALN format
MSA
http//www.ch.embnet.org/software/BOX_form.html
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Alignment Visualization Handeling
BoxShade
http//www.ch.embnet.org/software/BOX_form.html
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Outline
1. Pairwise Alignment

• 2. Getting Sequences MSA
• Option 1 Got Sequences Only MSA
• Option 2 BLAST then MSA
• Option 3 ConSeq Does It All

3. Alignment Visualization Handeling
4. Applications
36
Applications
Finding Motifs
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Applications
Conservation Analysis http//consurf.tau.ac.il
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Applications
Phylogenetic Trees
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MSA Tips

• The more the merrier but be careful...
• Unwanted sequences
• - Fragments- less than 40 the original
  sequence
• - Bad sequences- shot-gun projects ?
  unreliable sequences
• - Unrelated- examine identity between all
  sequences
• A blocky alignment is a good alignment
• Not too close, not too distant
• Define the usage of the alignment

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Thank You. mayas_at_bioinfo.tau.ac.il