Exercises Pairwise alignment Homology search (BLAST) Multiple alignment (CLUSTAL W) Iterative Profile Search: Profile Search –Pfam –Prosite –PSI-BLAST –SAM

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

Exercises OUT IN Cytc Fe Cu B Fe e-e- I e-e- e-e- O2O2 H2OH2O 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

Exercises H+H+H+H+ e-e- Nadh.dh succ.dh NADH e-e-e-e- e-e-e-e- succinate O2O2 H2OH2O O2O2 H2OH2O O2O2 H2OH2O e-e-e-e- cytochrome c oxidase ? ? quinol oxidase e-e- H+H+H+H+ cytbc 1 quinol e-e-e-e- Cytc Terminal Oxidases

Exercises Multiple Alignment

Exercises Multiple alignment: standard gap cost Multiple Alignment Ligands Cu center Ligands hemes Prosite pattern

Exercises Multiple alignment: large gap cost Multiple Alignment Ligands Cu center Prosite pattern Ligands hemes

Exercises Phylogenetic Tree Tree based on subselection

Exercises PROSITE

Exercises Prosite

Exercises Prosite

Exercises Prosite

Exercises Prosite

Exercises Prosite

Exercises

Prosite

Exercises Prosite domain Prosite

Exercises

Pattern & profile

Exercises

pFAM

Exercises Pfam

Exercises Pfam

Exercises Pfam

Exercises COX family Pfam

Exercises Pfam

Exercises Pfam

Exercises Pfam

Exercises Pfam

Exercises Pfam

Exercises Pfam

Exercises BLOCKS

Exercises Blocks

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

Exercises PSI-BLAST

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

Exercises PSI-BLAST

Exercises PSI-BLAST

Exercises PSI-BLAST

Exercises PSI-BLAST

Exercises PSI-BLAST

Exercises PSI-BLAST

Exercises SAM

Exercises SAM

Exercises SAM

Exercises SAM

Exercises

SAM Markov model Emission probability per AA Transition probabilities Insertion probability per AA position short.t2k-w0.5.mod

Exercises SAM input targets

Exercises SAM

Exercises SAM

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

Exercises Try to view the structure of the family SAM

Exercises SAM

Exercises SAM

Exercises Logos of the secondary structure prediction SAM

Exercises SAM

Exercises HMMer states Emission probability per AA Null model Transition probabilities Insertion probability per AA

Exercises