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HANDS-ON ConSurf! Web-Server: http://consurf.tau.ac.il/http://consurf.tau.ac.il/ The ConSurf webserver
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http://consurf.tau.ac.il/results/1290596232/output.php
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http://consurftest.tau.ac.il/ The ConSurf webserver
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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
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The ConSurf webserver
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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
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The ConSurf webserver
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Jmol- Easy web-based viewer
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View tree- phylogenetic relations
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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
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BioEdit http://www.mbio.ncsu.edu/BioEdit/bioedit.html
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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
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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…
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Viewing and producing images
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ConSurf for pymol The ConSurf webserver http://consurf.tau.ac.il/results/1290596232/output.php
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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
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Download! Producing images Press
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Producing images
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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
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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
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