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Lab 4.3

Lab 4.3. Comparative Modeling by M ODELLER. Roberto Sanchez Mount Sinai School of Medicine New York, NY, USA. roberto.sanchez@physbio.mssm.edu http://physbio.mssm.edu/~sanchez/. Comparative Modeling by M ODELLER. Using M ODELLER Example 1: Modeling of BLBP

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Lab 4.3

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  1. Lab 4.3 Comparative Modeling by MODELLER Roberto Sanchez Mount Sinai School of Medicine New York, NY, USA roberto.sanchez@physbio.mssm.edu http://physbio.mssm.edu/~sanchez/

  2. Comparative Modeling by MODELLER • Using MODELLER • Example 1: Modeling of BLBP • Example 2: Protein/ligand complex model • Example 3: Multiple template modeling

  3. Comparative Modeling by MODELLER • Obtaining MODELLER and related info • Using MODELLER • Example 1: Modeling of BLBP • Example 2: Protein/ligand complex model • Example 3: Multiple template modeling

  4. Obtaining MODELLER and related information • MODELLER web page http://guitar.rockefeller.edu/modeller/modeller.html • Download Software (UNIX/Linux) • HTML Manual • Join Mailing List

  5. Using MODELLER • No GUI!  • Controlled by command file (script)  • Script is written in TOP language  • TOP language is simple 

  6. Using MODELLER • INPUT: • Target Sequence (PIR format) • Template Structure (PDB format) • TOP command file • OUTPUT: • Target-Template Alignment • Model in PDB format • Other data

  7. EXAMPLES • Example files are in: /PATH/examples/ • Copy example files to your home directory with the following command: cp –R /PATH/examples .

  8. Example 1: Modeling of BLBPINPUT • Target: Brain lipid-binding protein (BLBP) • BLBP sequence in PIR (MODELLER) format, file examples/ex1/blbp.seq : >P1;blbp sequence:blbp:::::::: VDAFCATWKLTDSQNFDEYMKALGVGFATRQVGNVTKPTVIISQEGGKVVIRTQCTFKNTEINFQLGEEFEETSIDDRNCKSVVRLDGDKLIHVQKWDGKETNCTREIKDGKMVVTLTFGDIVAVRCYEKA* • Template search already done for you: • Template: PDB file 1HMS • examples/ex1/1hms.pdb

  9. Example 1: Modeling of BLBPSTEP 1: Align blbp and 1hms sequencesTOP script for target-template alignment File examples/ex1/align.top READ_MODEL FILE = '1hms.pdb' SEQUENCE_TO_ALI ALIGN_CODES = '1hms' READ_ALIGNMENT FILE = 'blbp.seq', ALIGN_CODES = 'blbp', ADD_SEQUENCE = on ALIGN WRITE_ALIGNMENT FILE='blbp-1hms.ali', ALIGNMENT_FORMAT = 'PIR' WRITE_ALIGNMENT FILE='blbp-1hms.pap', ALIGNMENT_FORMAT = 'PAP' Run by typing mod align.top in the examples/ex1/ directory. If something goes wrong check file align.log

  10. Example 1: Modeling of BLBPSTEP 1: Align blbp and 1hms sequences OUTPUT File examples/ex1/blbp-1hms.ali >P1;1hms structureX:1hms: 1 : : 131 : :undefined:undefined:-1.00:-1.00 VDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTFKNTEISFKLGVEFDETTA DDRKVKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLILTLTHGTAVCTRTYEKE* >P1;blbp sequence:blbp: : : : : : : 0.00: 0.00 VDAFCATWKLTDSQNFDEYMKALGVGFATRQVGNVTKPTVIISQEGGKVVIRTQCTFKNTEINFQLGEEFEETSI DDRNCKSVVRLDGDKLIHVQKWDGKETNCTREIKDGKMVVTLTFGDIVAVRCYEKA*

  11. Example 1: Modeling of BLBPSTEP 1: Align blbp and 1hms sequences OUTPUT File examples/ex1/blbp-1hms.pap _aln.pos 10 20 30 40 50 60 1hms VDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTFKNT blbp VDAFCATWKLTDSQNFDEYMKALGVGFATRQVGNVTKPTVIISQEGGKVVIRTQCTFKNT _consrvd **** **** ** *** *** ********** **** ** * * ***** _aln.pos 70 80 90 100 110 120 1hms EISFKLGVEFDETTADDRKVKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLILTLTHG blbp EINFQLGEEFEETSIDDRNCKSVVRLDGDKLIHVQKWDGKETNCTREIKDGKMVVTLTFG _consrvd ** * ** ** ** *** ** * *** ** * ***** ** ** *** *** * _aln.pos 130 1hms TAVCTRTYEKE blbp DIVAVRCYEKA _consrvd * * ***

  12. Example 1: Modeling of BLBPSTEP 2:Model the blbp structure using the alignment from step 1. TOP script for model building File examples/ex1/model.top INCLUDE SET ALNFILE = 'blbp-1hms.ali' SET KNOWNS = '1hms' SET SEQUENCE = 'blbp' SET STARTING_MODEL = 1 SET ENDING_MODEL = 1 CALL ROUTINE = 'model' Run by typing mod model.top in the examples/ex1/ directory. If something goes wrong (or just for fun) check file model.log

  13. Example 1: Modeling of BLBPSTEP 2:Model the blbp structure using the alignment from step 1. OUTPUT File examples/ex1/blbp.B99990001 • PDB file • Can be viewed with Rasmol, PDBviewer, etc. • Transfer from SGI server to NT workstation using ftp: • ftp server • cd examples/ex1/ • get blbp.B99990001

  14. Example 2: Modeling of a BLBP/Oleic Acid complexINPUT • BLBP – 1hms alignment from Example 1 cd examples/ex2 cp ../ex1/blbp-1hms.ali blbp-1hms-ola.ali >P1;1hms structureX:1hms: 1 : : 133 : :undefined:undefined:-1.00:-1.00 VDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIEKNGDILTLKTHSTFKNTEISFKLGVEFDETTA DDRKVKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLILTLTHGTAVCTRTYEKE.* >P1;blbp sequence:blbp: : : : : : : 0.00: 0.00 VDAFCATWKLTDSQNFDEYMKALGVGFATRQVGNVTKPTVIISQEGGKVVIRTQCTFKNTEINFQLGEEFEETSI DDRNCKSVVRLDGDKLIHVQKWDGKETNCTREIKDGKMVVTLTFGDIVAVRCYEKA.* Oleic acid molecule in 1hms PDB file is residue number 133. This can be checked by RASMOL or by looking at the PDB file directly.

  15. Example 2: Modeling of a BLBP/Oleic Acid complexTOP script for modeling File examples/ex2/model2.top INCLUDE SET ALNFILE = 'blbp-1hms-ola.ali' SET KNOWNS = '1hms' SET SEQUENCE = 'blbp' SET STARTING_MODEL = 1 SET ENDING_MODEL = 1 SET HETATM_IO = on CALL ROUTINE = 'model' Run by typing mod model2.top in the examples/ex2/ directory. If something goes wrong check file model2.log

  16. Example 2: Modeling of a BLBP/Oleic Acid complex.OUTPUT File examples/ex2/blbp.B99990001 • View ligand with rasmol: • Menu: • Display -> backbone • Colours -> structure • Command line: • RasMol> select ligand • RasMol> color green • RasMol> cpk

  17. Example 3: Multiple template modeling INPUT • Target: Brain lipid-binding protein (BLBP) • BLBP sequence in PIR (MODELLER) format, file examples/ex3/blbp.seq : • Templates: PDB files 1HMS and • examples/ex3/1hms.pdb • examples/ex3/1lid.pdb

  18. Example 3: Multiple template modelingSTEP 1: Align blbp and template sequencesTOP script for target-template alignment File examples/ex3/align.top SET ALIGN_CODES = '1hms''1lid' SET ATOM_FILES = '1hms.pdb' '1lid.pdb' MALIGN3D SET ADD_SEQUENCE = on, ALIGN_BLOCK = 2 READ_ALIGNMENT FILE = 'blbp.seq', ALIGN_CODES = 'blbp' ALIGN WRITE_ALIGNMENT FILE = 'blbp-1hms-1lid.ali' WRITE_ALIGNMENT FILE = 'blbp-1hms-1lid.pap', ALIGNMENT_FORMAT = 'PAP' Run by typing mod align.top in the examples/ex3/ directory. If something goes wrong check file align.log

  19. Example 3: Multiple template modelingSTEP 2:Model the blbp structure using the alignment from step 1. TOP script for model building File examples/ex3/model3.top INCLUDE SET ALNFILE = 'blbp-1hms-1lid.ali' SET KNOWNS = '1hms' '1lid' SET SEQUENCE = 'blbp' SET STARTING_MODEL = 1 SET ENDING_MODEL = 1 CALL ROUTINE = 'model' Run by typing mod model3.top in the examples/ex3/ directory. If something goes wrong check file model3.log

  20. Model your own protein • Template Search (BLAST) http://www.ncbi.nlm.nih.gov/blast/ • Model Evaluation (VERIFY3D) http://www.doe-mbi.ucla.edu/verify3d.html

  21. VERIFY3D Alignment with errors Correct Alignment

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