1 LSM2241 AY0910 Semester 2 MiniProject Briefing Round 5

2 FOCUS Identify and analyze sequence differences among NF-κB protein sequences

Specific objectives 1.Analyze mammalian NF-κB proteins and their homologues 2.Generate sequence patterns for conserved regions in mammalian NF-κB proteins and their homologues 3.Map conserved sequence patterns to 3D structures

Topics for Round 5 Data collection - DONE Data processing - DONE Data analysis –Phylogenetic analysis Multiple Sequence Alignment - DONE Generating a phylogenetic tree - DONE –Sequence pattern analysis – TODAY –Structural analysis 4

Round 4 Review Adding root sequence to your MSA Editing your MSA Generating a phylogenetic tree with bootstrap values Any questions? 5

Sequence Pattern Analysis Goal To identify patterns within conserved domains of NF-κB proteins How? Generate Prosite patterns within the conserved domains of selected mammalian NF-κB proteins using the PRATT tool 6

Identifying highly conserved sequences from your phylogenetic tree From the phylogenetic tree generated in the previous section, record the accession numbers of the sequences that are present in each statistically significant clade (bootstrap value ≥ 70) Note: Record the accession numbers for each clade separately 7

Identifying highly conserved sequences from your phylogenetic tree 8

Extract the aligned conserved domain sequences for each statistically significant clade from the edited multiple sequence alignment file Launch BioEdit: Edit  Search  Search for  titles that contain a list of substrings In the popup window, type or paste the list of accession numbers you want to extract in the blank field and click “OK” Note: Do this separately for each statistically significant clade 9

Identifying highly conserved sequences from your phylogenetic tree 10

Identifying highly conserved sequences from your phylogenetic tree 11

Identifying highly conserved sequences from your phylogenetic tree Now, all the sequences containing the accession numbers you have keyed in will be selected. Copy these sequences by selecting Edit  Copy Sequence(s). Open a blank alignment file (File  New Alignment) and paste your sequences into the blank alignment file (Edit  Paste) Save your new alignment in FASTA format Repeat for sequences in each statistically significant clade 12

Generating Patterns Do you think you need to do some processing to the alignment of each clade before generating a pattern? 13 Original sequence Clade alignment new slide

Generating Prosite patterns Use the PRATT tool to generate Prosite patterns for parts of each statistically significant clade (follow the instructions in P7) Scan your patterns against all mammalian NF-κB sequences –How do you obtain these sequences? 14 slide updated

Generating Prosite patterns 15 Copy and paste the contents of nfkb_all.fas in the ScanProsite page, under “Sequence(s) to be scanned”

Generating Prosite patterns Analyze your results: –Compare and contrast each Prosite pattern. How are they similar or different? –Compare the results for each Prosite pattern. How many hits do you get for each Prosite pattern? –Which are the proteins that contain your pattern? –How accurate is your pattern? –Are your results reliable? If not, what can you do to generate more reliable results? 16

Generating Prosite patterns Repeat the ScanProsite step again This time, scan your patterns against UniprotKB/Swiss-Prot, UniProtKB/TrEMBL and PDB. Select the option: include all splice variants. 17

Generating Prosite patterns Compare and contrast your results –How many hits do you get now? –Do you get the same results as the last step? Why? 18

Generating Prosite patterns Analyze your results: –Compare and contrast each Prosite pattern. How are they similar or different? –Compare the results for each Prosite pattern. How many hits do you get for each Prosite pattern? –Which are the proteins that contain your pattern? –How accurate is your pattern? –Are your results reliable? If not, what can you do to generate more reliable results? 19

Generating Prosite patterns Points for discussion –What is the significance of sequence pattern analysis? In what ways are Prosite patterns useful? –What is the significance of your results? –Are there any other pattern generation software? Do they generate better results? –How can you improve on your results? 20

Topics for Round 5 Data collection - DONE Data processing - DONE Data analysis –Phylogenetic analysis Multiple Sequence Alignment - DONE Generating a phylogenetic tree - DONE –Sequence pattern analysis - DONE –Structural analysis – NEXT WEEK 21