1. 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/

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

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

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

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

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

7. Using M ODELLER Installed on the SUN server telnet to the server Before you run M ODELLER type: source mod-setup (You can also put this into your.cshrc file) To run M ODELLER type: mod script.top

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

9. Example 1: Modeling of BLBP INPUT Target: Brain lipid-binding protein (BLBP) BLBP sequence in PIR (M ODELLER ) format, file examples/ex1/blbp.seq : >P1;blbp sequence:blbp:::::::: VDAFCATWKLTDSQNFDEYMKALGVGFATRQVGNVTKPTVIISQEGGKVVIRTQCTFKNTEINFQLGEEFEETSI DDRNCKSVVRLDGDKLIHVQKWDGKETNCTREIKDGKMVVTLTFGDIVAVRCYEKA* Template search already done for you: Template: PDB file 1HMS examples/ex1/1hms.pdb

10. Example 1: Modeling of BLBP STEP 1: Align blbp and 1hms sequences TOP 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

11. Example 1: Modeling of BLBP STEP 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*

12. Example 1: Modeling of BLBP STEP 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 * * ***

13. Example 1: Modeling of BLBP STEP 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

14. Example 1: Modeling of BLBP STEP 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

15. Example 2: Modeling of a BLBP/Oleic Acid complex INPUT 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 R ASMOL or by looking at the PDB file directly.

16. Example 2: Modeling of a BLBP/Oleic Acid complex TOP script for modeling File examples/ex2/model2.top Run by typing mod model2.top in the examples/ex2/ directory. If something goes wrong check file model2.log 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'

17. 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

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

19. Example 3: Multiple template modeling STEP 1: Align blbp and template sequences TOP 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

20. Example 3: Multiple template modeling STEP 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

21. 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

22. VERIFY3D Alignment with errorsCorrect Alignment