1. Checkout trunk/antibody 2. Change ~/antibody/scripts/rampaths.txt to set the paths to match your directory structure 3. Copy the following files to directory where simulation will be run: i. ~/antibody/scripts/rampaths.txt ii. ~/antibody/scripts/utilities.txt 4. Have the following files ready for input in the simulation directory: i. query_l.fasta: sequence of light chain (not required for camelid VHH antibody) ii. query_h.fasta: sequence of heavy chain Note: The fasta files should conform to the following format: a. The header line with > should not be there b. There should not be any hyphens for breaks c. There should not be any newline charchter - all on one line 5. Make sure that Profit is installed on your system and it is set up such that it can be called from any subfolder by typing "profit". 6. Change the paths.txt in ~/antibody/scripts/paths.txt such that in all cases it points to your local copy of the rosetta_database directory 7. Change the hardcoded paths in the file ~/antibody/scripts/ram_blankcdrs.pl They should point to two files located in ~/antibody/scripts/. Make sure that the full absolute paths (non-relative) are provided. For some strange reason incorporating rampaths.txt does not work here, more details are provided in ram_blankcdrs.pl 8. Change the hardcoded paths in the following file to match your directory structure: ~/antibody/scripts/get_top10_scoring.pl 9. Also ensure that you have the most updated version of the rosetta_database directory 10. Change paths for sam/jufo/blosum etc in the following file: ~antibody/scripts/make_rosetta_fragments.pl 11.In the folder in which simulations will be run,which already contains: i. rampaths.txt ii. utilities.txt iii. query_l.fasta (not present for camelid VHH antibody) iv. quary_h.fasta Launch the following command for classical antibody: perl -P ~/antibody/scripts/ram_buildloop_wrapper.pl pdb1xyz_chothia.pdb bit 1 1 1 1 1 1 1 2000 query_l.fasta query_h.fasta Launch the following command for camelid VHH antibody: perl -P ~/antibody/scripts/ram_buildloop_wrapper.pl pdb1xyz_chothia.pdb bit 1 1 1 1 1 1 1 2000 query_l.fasta query_h.fasta camelid 12.The simulations folder will have a new directory named "build". Some of the important files in the directory are: i. FR02.pdb: The framework region with the non-H3 CDRs grafted in ii. H3_CTERM: Fragment files for the CDR H3 base region iii. aaFR02_03_05.200_v1_3: 3-mer fragment file for the H3 loop region with antibody fragments appended iv. aaFR02_09_05.200_v1_3: 9-mer standard rosetta fragment v. lfr.pdb: light chain template from which light chain of FR02.pdb has been obtained (not present for camelid VHH antibody) vi. hfr.pdb: heavy chain template from which heavy chain of FR02.pdb has been obtained vii. 1xyz_build_loops.con: The condor script file containing headers. This is used for launching jobs on the cluster using the command: "condor_submit 1xyz_build_loops.con" viii.1xyz_build_loops.bash: This is called by the .con file (above) and contains the actual rosetta command line needed for launching a job with 2000 decoys. 13.Post processing: In the directory one level above the "build" directory launch the script: ~/antibody/scripts/get_top10_scoring.pl The top ten models are outputted: model1.pdb.....model10.pdb The processed scorefile void of any redundancies and containing decoys which do not have broken CDR H3s is also outputted as: aaFR02_unbroken.fasc

UPDATING THE ANTIBODY DATABASE: Use the script: ~/antibody/scripts/update_RosettaAntibody_database.pl (follow instructions in the script)

References: For camelid VHH antibody modeling: Aroop Sircar, Kayode A. Sanni, Jiye Shi & Jeffrey J. Gray, "Analysis and Modeling of the Variable Region of Camelid Single Domain Antibodies," J. Immunology, 186(11), 6357-6367 (2011).

For classical Fv modeling: Arvind Sivasubramanian*, Aroop Sircar*, Sidhartha Chaudhury & Jeffrey J. Gray, "Toward high-resolution homology modeling of antibody Fv regions and application to antibody-antigen docking," Proteins: Structure, Function and Bioinformatics, 74(2), 497-514 (2009).