Aligning Kinases Applying MSA Analysis to the CDK family

Building A Multiple Sequence Alignment

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1 Organizing a Family Gathering The CDK example

Choosing the Right Sequences SwisProt Litterature Other Databases

Organizing the Data SRS Public Data IGS Data Aventis CDK Genecard Manual Automatic

Accessing the Data: The Fischer Server Fischer will Contain – A collection of Flat files – A secure SRS server – File Formats The server is a Technology Pipeline – Can be adapted in real time – Can be Transfered

Our CDK Data CDKs and CDK-like – Protein Information Functional Features Structural Information – Genomic Information Genes Variant SNPs

Our MSA dataset 29 amino acid sequences (CDKS and Aurora families, stemming from primary transcripts) – 2 isoforms of a cdk member 4 PDB structures : – 1MUO (AUR A) – 1BLX (CDK 6 ) – 1b38 (CDK 2) – 1H4L (CDK 5) Use of T-coffee release 1.78 with integration of the structure informations contained in pdb files

2 Aligning The Sequences

Building A Multiple Sequence Alignment ClustalW T-Coffee Muscle Hand Editing Combination Comparison

Using Structural Information 3D-Coffee Struct Vs Struct Seq Vs Struct Thread Superpose Seq Vs Seq Local Global

Method

Accessing the Methods: Fischer Public 3D-Coffee server – igs-server.cnrs-mrs.fr/TCoffee/ Fischer – Latest version of T-Coffee – Customised parameters – Coktails of MSA methods

3 Dressing Up a Multiple Sequence Alignment

Feature Dressing -25 Binding site -20 Phospho -40 nsSNP -50 Splice Site … Escript

Feature Dressing

4 How Good Is The Alignment ????

T-Coffee CORE Evaluation

CORE index Specificity (  ) and Sensitivity (  )

Feature Based Evaluation

Features mapping on multiple alignment T-coffee ATP binding site Glycine loop ATP binding site Glycine loop Non-synonymous SNP ClustalW

Structure Based Evaluation APDB

Include Sequences with Known Structures – Do Not use Structural Information Score 1 – Use Structural Information:Score 2 If Score1 ~ Score 2 – Structural Information does not help much – The alignment is of reasonnable quality

Evaluating a Multiple Sequence Alignment T-Coffee CORE index Feature Based Library APDB

Maninupulating and Comparing Alignments Reformating/Processing – seq_reformat – extract_from_pdb Coloring – seq_reformat – ESCript Comparing – aln_compare

5 Thinking Large ????

T-Coffee_dpa T-Coffee is limited to a small number of sequences T-coffee_dpa: Double Progressive Algo – Able to handle large datasets – 1000 sequences and more – Able to use structural information

Using A Multiple Sequence Alignment

1 Exploring The Alignment

Cdk's signature Cdk's T-loop (orange) and aurora's Activating loop Substrat recognition motif

2 Using The Alignment Does my Sequence Make Sense

Identifying Abnormalities within an MSA Insertion within the Nuc Binding Site…

Identifying Abnormalities within an MSA

Activation loop (orange)

Identifying Abnormalities within an MSA Retinoblastoma

2 Using The Alignment Analysing the Structure with The Alignment

The Evoltionnary Trace

3 Using The Alignment Spotting differences

What makes a CDK not and AurorA

4 Clustering and Correlating

Function Trees Vs Lead Trees 1-Select Functionnaly Important Positions 2-Make a tree based on these positions 3-Compare the tree with the lead tree PROBLEMS: – Choose on the right positions – Describe the Leads with the right determinants