1. HANDS-ON ConSurf! Web-Server: http://consurf.tau.ac.il/http://consurf.tau.ac.il/ The ConSurf webserver

2. http://consurf.tau.ac.il/results/1290596232/output.php

3. http://consurftest.tau.ac.il/ The ConSurf webserver

4. http://consurftest.tau.ac.il/ The ConSurf webserver Choose parameters for BLAST search & MSA: Database- uniref90 recommended, or uniprot Muscle\MAFFT MSA method, others are also fine Number of homologues- 500 recommended Minimum identity- 20-25% PSI-BLAST iteration: 1-2, but start with 1 Evalue- ~0.0001

5. The ConSurf webserver

7. MSA colored by conservation Grades file ConSurf for pymol BLAST & sequences MSA- download and view with BioEdit Phylogenetic tree The ConSurf webserver http://consurf.tau.ac.il/results/1290596232/output.php

8. The ConSurf webserver

9. Jmol- Easy web-based viewer

10. View tree- phylogenetic relations

11. BioEdit MSAs cannot be easily examined without an appropriate program The programs enable easy viewing, coloring, presenting and editing of MSAs consisting of a large number of sequences. Some viewers are also able of running other programs, e.g. performing an alignment when sequences are given as input. Two of the top viewers are: BioEdit- briefly presented next JalView

12. BioEdit http://www.mbio.ncsu.edu/BioEdit/bioedit.html

13. Find a specific sequence: “Edit-> search -> in titles” Erase\add sequences: “Edit-> cut\paste\delete sequence” “Sequence Identity matrix” under “Alignment”- useful for a rough evaluation of distances within the alignment. After taking out sequences, “Minimize Alignment” under “Alignment” takes out unessential gaps. Can save an image using: “File -> Graphic View” & then “Edit -> Copy page as BITMAP” BioEdit

14. A good MSA has: Blocks corresponding to SSE elements, e.g. the helices and the betas sheets Regions with more gaps of variable lengths- loop regions Not too close, not to far… learned from experience…

15. Viewing and producing images

16. ConSurf for pymol The ConSurf webserver http://consurf.tau.ac.il/results/1290596232/output.php

17. High quality via PyMol Open the PDB file downloaded from the PyMol instructions (“File  Open…”) Open the script “consurf_new.py“ downloaded from the PyMol instructions (“File  Run…”) Write “colour_consurf” in the command line Create the image “ray [x] [y]” in the command line Save the image “png [image name], dpi=300” Producing images

18. Download! Producing images Press

19. Producing images

24. 1.Go to the result page. 2.Examine the outputs described above. 3.Download the structure with conservation scores for pymol, show it colored by conservation in pymol. 4.Create a nice picture. Exercise: NhaA, PDBid 1ZCD, chain A http://consurf.tau.ac.il/results/1290328145/output.php

25. Bottom lines Many homologs needed! Minimum a few dozens The Quality of the MSA completely determines quality of conservation analysis  Trial & error: various parameters for homolog collection & MSA building- BLAST database, number of rounds, Evalue…. Examine each alignment in BioEdit With or without structure: insight into functional vs. structurally important positions