Assess the impact of mutations on antibody-antigen binding affinity
OpenMM: 7.3.1
OpenMMTools: 0.16.1
CUDA: 8.0
clean_pdb_rerefined.ipynb, clean_pdb_4jha.ipynb, edit_seqres.ipynb, split_chain_and_cap.ipynb
- Preprocess SEQRES -- Use edit_seqres.ipynb to programatically split delete lines and/or split chains in the SEQRES.
- Description of edit_seqres.ipynb: This script calls 3 functions to manipulate a SEQRES: 1) delete lines (or split_chains) 2) fill SEQRES lines that are not full (13 residues) 3) reindex SEQRES lines so that indexes are consecutive. Output: PDB file with desired SEQRES sequence removed
- Split F protein chain -- Use split_chain.ipynb
- Description of split_chain.ipynb: This script uses the OpenMM Topology object to create a new topology with the F protein chain(s) split into two (because it contains a long missing loop that was not added back in). Terminal atoms added to ends of chains using PDBFixer.
- Manually copy CRYST1 line and the edited SEQRES to the PDB with the F protein split.
- Clean PDB -- Use clean_pdb.ipynb
- Description of clean_pdb.ipynb: This script uses PDBFixer to add missing residues and remove heterogens. Note: If the missing residues were part of a long terminal fragment (i.e. > 10 residues), then they will not be added them back in.
- Add CRYST1 line from the original PDB if the final PDB doesn't contain it.
data/4jhw/4jhw.pdb and data/5udc/5udc.pdb
Note: the files for each structure are in order from input to output and accumulate changes, e.g. 4jhw_noloop_noseqres.pdb contains changes from 4jhw_noloop.pdb)
5udc
- data/5udc/5udc.pdb - raw PDB
- data/5udc/5udc_noloop.pdb - deleted residues that are not present in 4jhw, 4jha, 5k6f
- data/5udc/5udc_noloop_seqressplit.pdb - split chain in SEQRES near missing loop that will not be added back in
- data/5udc/5udc_splitchain.pdb - F protein chain split into two chains
- data/5udc/5udc_splitchain_capped.pdb - terminal atoms added to ends of chains using PDBFixer
- data/5udc/5udc_clean.pdb — missing residues not in long (> 10 residues) terminal fragments added back in
4jhw
- data/4jhw/4jhw.pdb — raw PDB
- data/4jhw/4jhw_noloop.pdb - deleted residues that are not present in 5udc, 4jha, 5k6f
- data/4jhw/4jhw_noloop_seqressplit.pdb - split chain in SEQRES near missing loop that will not be added back in
- data/4jhw/4jhw_splitchain.pdb - F protein chain split into two chains
- data/4jhw/4jhw_splitchain_capped.pdb - terminal atoms added to ends of chains using PDBFixer
- data/4jhw/4jhw_clean.pdb — missing residues not in long (> 10 residues) terminal fragments added back in
4jhw
- Load 4jhw.pdb into edit_seqres.ipynb to delete residues and split chain in the SEQRES near missing loop that will not be added back in. Write this to 4jhw_noloop.pdb (after deleting residues), then 4jhw_noloop_seqressplit.pdb.
- Load 4jhw.pdb into split_chain.ipynb. Use the split_chain function to save topology and positions with chain split to 4jhw_splitchain.pdb. Then use the cap_chain function to save topology and positions with missing terminal atoms added to 4jhw_splitchain_capped.pdb.
- Manually copy the SEQRES from 4jhw_noloop_seqressplit.pdb to the top of 4jhw_splitchain_capped.pdb.
- Load 4jhw_splitchain_capped.pdb into clean_pdb.ipynb. Follow PDBFixer protocol (findMissingResidues(), remove terminal missing fragments from fixer.missingResidues, findNonStandardResidues(), removeHeterogens, findMissingAtoms(), addMissingAtoms()). Write to 4jhw_clean.pdb.
For this structure, remove the 99-109 region (and the linker) and then split the chain at this region, using split_chain_and_cap.ipynb. There are no missing residues so no need to clean with PDBFixer.
For this structure, add missing residues based on 4jhw antibody using clean_4jha.ipynb. There is no need to split chains here.
- Determine antibody with best electron density and use UCSF Chimera to remove chains from the unwanted antibodies.
- Add CRYST1 line from the original PDB if the final PDB doesn't contain it
- Copy 4jhw_final_v2_refmac1_clean.pdb to new file 4jhw_final_v2_refmac1_clean_trimer.pdb.
- Load 4jhw_final_v2_refmac1_clean_trimer.pdb as "Biological Assembly" in UCSF Chimera
- Render biological assembly: Model panel > biological unit
- Combine the assembly into one model: Copy/combine
- Save the PDB: File > Save PDB
- Remove chains of unwanted antibodies in Chimera
- Change chain ids to match 5udc trimer F protein chains: Tools > Structure editing > Change chain IDs
- Save the PDB: File > Save PDB (renumbered/holo/nonoverlay/4jhw_final_v2_refmac1_clean_trimer_single_ab.pdb)
- Add CRYST1 line from the original PDB if the final PDB doesn't contain it
delete_residues.ipynb
This script deletes residues at the n-terminus or c-terminus as specified by the user. It caps the chain at the terminus with deleted residues.
- Load PDBs into UCSF Chimera
- File > Open > Select PDB file
- Overlay
- Tools > Structure comparison > Matchmaker (Use default settings)
- Delete low res reference chains
- Select > Chain
- Actions > Atoms/Bonds > Delete
- Merge models
- Tools > General controls > Model panel > Select the two models, choose “copy/combine"
- Change Chain IDs so that they correspond to chain IDs in the low res holo structure
- Tools > Structure editing > Change chain IDs
- File > Save PDB
- Save with respect to low res structure
- Load PDB into UCSF Chimera
- File > Open > Select PDB file
- Delete all chains for two of the monomers
- Select > Chain
- Actions > Atoms/Bonds > Delete
- Note: To select multiple chains at a time (e.g. chains F through L), enter into Chimera's command line:
select :.f-l - Note: I kept H, L, F chains in the original PDB (aka chains A-D in the cleaned PDB) because these required adding the least number of missing loop residues
- File > Save PDB
Note: To extract the F protein or antibody, follow this same procedure, but select the relevant chains.
run_em.py, run_em.sh, remove_solvent_from_traj.py, join_restarted.py
(to be run on lilac)
- Run
module load cuda/9.2 - Activate conda environment
- Navigate to the directory containing run_em.py and run_em.sh and run
bsub < run_em.sh - After jobs have completed, remove solvent from trajectories using remove_solvent_from_traj.py
- If there were jobs that were restarted, join the original and restarted trajectories and CSVs using join_restarted.py
restart_em.py, restart_em.sh
- Run
module load cuda/9.2 - Activate conda environment
- Navigate to the directory containing restart_em.py and restart_em.sh and run
bsub < restart_em.sh - After jobs have completed, remove solvent from trajectories using remove_solvent_from_traj.py
analyze_em.py, analyze_em_interface_residues.py, analyze_em_interface_rmsd.py
- Run analyze_em.py to create plots of state data (PE, KE, T, V) and RMSD (after aligning on whole molecule).
- Run analyze_em_interface_residues.py to create dataframes of interface residues in each chain.
- Run analyze_em_interface_rmsd.py to plot RMSDs (interface, F protein, antibody) after aligning different components (interface, F protein, antibody).
(In PyMOL)
Rafal’s tips:
- Display > Quality > Maximum performance
- Display > Color Space > CMYK
- set ray_trace_mode, 3
- show spheres
- set sphere_transparency, 0.85
- set movie_fps, 60
- smooth
- zoom
- Center it on the right view of the interface
- Smooth (can do this multiple times, will remove the faster movements more and more)
- Save as pymol sessions (if need to align complexes in different movies, can just load other complex into this session and then superpose/align the other complex then save as movie)
- File > Export as movie.. MPEG (make the window as big of a square as possible —> 1600x1600 for high quality)