Exercises Exercises

(1)

Exercises

Exercises

• Pairwise alignment

• Homology search (BLAST)

• Multiple alignment (CLUSTAL W)

• Iterative Profile Search:

• Profile Search – Pfam

– Prosite – PSI-BLAST – SAM

(2)

Exercises

Overview

Query Sequence Unknown

Blast Sequence to search for close homologs

Search pFAM, Prosite for conserved motifs

You detected homology with an annotated

protein family

Make a multiple sequence alignment Generate profile or HMM

Search database for remote homologs Blast

ClustalW PFAM

PROSITE

HMMer, PSSM

Profile Search

PSI-blast

(3)

Exercises

OUT

IN Cytc

Fe FeCu_B e^-

I Fe Fe e^-

e^-

O₂ H₂O Fe

Terminal Oxidases

• Unknown protein is a heme cupper oxidase

• Enzyme that reduces O2 to H2O in respiratory chain

• Subunit contains 2 hemes and a Cu prosthetic group

• The residues that are ligands of these groups have been conserved in all types of terminal oxidase complexes

(4)

Exercises

HH⁺⁺ e

-

Nadh.dh succ.dh NADH

e e

^-^-

e e

^-^-

succinate

O₂ H₂O

e e

^-^-

cytochrome

cytochrome c oxidasec oxidase

?

quinol oxidase quinol oxidase

e^-

HH⁺⁺

cytbc₁

quinol

e e

^-^-

Cytc

Terminal Oxidases

(5)

Exercises

Multiple Alignment

(6)

Exercises

Multiple alignment: standard gap cost

Multiple Alignment

Ligands Cu center

Ligands hemes

Prosite pattern

(7)

Exercises

Multiple alignment: large gap cost

Multiple Alignment

Ligands Cu center

Prosite pattern

Ligands hemes

(8)

Exercises

Phylogenetic Tree

Tree based on subselection

(9)

Exercises

PROSITE

(10)

Exercises

Prosite

(11)

Exercises

Prosite

(12)

Exercises

Prosite

(13)

Exercises

Prosite

(14)

Exercises

Prosite

(15)

Exercises

(16)

Exercises

(17)

Exercises

Prosite

(18)

Exercises

Prosite domain

Prosite

(19)

Exercises

(20)

Exercises

Pattern & profile

(21)

Exercises

(22)

Exercises

(23)

Exercises

(24)

Exercises

pFAM

(25)

Exercises

Pfam

(26)

Exercises

Pfam

(27)

Exercises

Pfam

(28)

Exercises

COX family

Pfam

(29)

Exercises

Pfam

(30)

Exercises

Pfam

(31)

Exercises

Pfam

(32)

Exercises

Pfam

(33)

Exercises

Pfam

(34)

Exercises

Pfam

(35)

Exercises

BLOCKS

(36)

Exercises

• Blocks

(37)

Exercises

(38)

Exercises

(39)

Exercises

Overview

Query Sequence Unknown

Blast Sequence to search for close homologs

Search pFAM, Prosite for conserved motifs

You detected homology with an annotated

protein family

Make a multiple sequence alignment Generate profile or HMM

Search database for remote homologs Blast

PFAM PROSITE

HMMer, PSSM

Profile Search

PSI-blast

(40)

Exercises

PSI-BLAST

(41)

Exercises

• PSI BLAST

– Start from a single sequence – Blast it against NCBI

– Select high scoring hits – Perform multiple alignment – Construct profile

– Iterate and find remote homologs

• Usually cut the sequence in pieces

• Avoid to give as input multi domain proteins

PSI-BLAST

(42)

Exercises

PSI-BLAST

(43)

Exercises

PSI-BLAST

(44)

Exercises

PSI-BLAST

(45)

Exercises

PSI-BLAST

(46)

Exercises

PSI-BLAST

(47)

Exercises

PSI-BLAST

(48)

Exercises

SAM

(49)

Exercises

SAM

http://www.cse.ucsc.edu/research/compbio/HMM-apps/HMM-applications.html

(50)

Exercises

SAM

(51)

Exercises

SAM

(52)

Exercises

(53)

Exercises

SAM Markov model

Emission probability per AA Transition probabilities

Insertion probability per AA position

short.t2k-w0.5.mod

(54)

Exercises

SAM

input

targets

(55)

Exercises

SAM

(56)

Exercises

SAM

(57)

Exercises

SAM

Hit with highest score! Hit with a protein family for which the 3D structure has been determined

(58)

Exercises

Try to view the structure of the family

SAM

(59)

Exercises

SAM

(60)

Exercises

SAM

(61)

Exercises

Logos of the secondary structure prediction

SAM

(62)

Exercises

SAM

(63)

Exercises

HMMer

states

Emission probability per AA Null model

Transition probabilities

Insertion probability per AA

(64)

Exercises

(65)

Exercises

(66)

Exercises

(67)

Exercises

(68)

Exercises