CRISPro

An Automated Pipeline for Protein Conformation Stabilization by Proline

Motivation

Recent studies have shown that the yield, antigenicity, and immunogenicity of an immunogen can be enhanced by stabilizing it into a specific conformation. Such stabilization often involves the engineering of proline mutations at residue positions where a proline is structurally compatible with the target conformation, but not with the alternative conformation. However, there is no publicly available tool that can design proline mutations for this purpose automatically. Here, we implemented an automated tool, CRISP, that inputs structural coordinates of a target conformation and/or an alternative conformation, and outputs a list of residue positions where proline mutations are predicted to stabilize the target conformation based on compatibility of phi-psi angles, secondary structure, and steric clashes. Thus, CRISP can be used to engineer immunogens into specific conformation and to design serologic probes, capable of isolating antibodies that recognize a target shape.

Installation

CRISPro has been successfully tested on Linux systems

Environment

CRISPro is best installed in CONDA environment using the following procedure:

• Download and install conda environment for 64-bit linux (https://conda.io/miniconda.html) using Python 2.7
  • Download: Miniconda2-latest-Linux-x86_64.sh
  • Install in command line: ./Miniconda2-latest-Linux-x86_64.sh (Add conda path to ~/.bashrc)
  • Open a new terminal window or source your bash with: source ~/.bashrc
• Create CRISPro environment in command line: conda create --name CRISPro
• Activate CRISPro environment in command line: conda activate CRISPro
• Install require packages by running the following in command line:

  • conda install -c r r

  • conda install -c conda-forge r-bio3d

  • conda install -c r r-foreach

  • conda install -c r r-doparallel

  • conda install -c samoturk pymol

  • sudo apt-get install dssp

Run

CRISPro can be run in the command line

Several input arguments are necessary to run the CRISPro.

1. Overlap cutoff, 1.5 is recommended
2. Phi-Psi cutoff, 0.01 is recommended
3. Number of cores
4. Path to file containing trans-proline phi-psi angles
5. Path to PyMOL script that mutates positions
6. Output prefix, for instance "OUTPUT"
7. Option (either 1, 2, or 3)
8. PDB file of target conformation (https://www.rcsb.org/pdb/static.do?p=file_formats/pdb/index.html) (Option 1 and 2)
9. Chain within PDB file of desired conformation (Option 1 and 2)
10. PDB file of alternative conformation (https://www.rcsb.org/pdb/static.do?p=file_formats/pdb/index.html) (Option 1 and 3)
11. Chain within PDB file of alternative conformation (Option 1 and 3)

Execute in the command line

R --vanilla < CRISPro_v.1.1-6.R 1.5 0.01 8 rama8000-transpro.data mutate.py OUTPUT 1 target.pdb A alternative.pdb A

Result

• Results will be saved in OUTPUT_DF.csv
• Explanation of Results
  • Option 1 (with target and alternative conformation input) columns include:
    • Residue number
    • Chain in target conformation
    • Amino acid in target conformation
    • Phi angle in target conformation
    • Psi angle in target conformation
    • Compatibility of trans proline angles in target conformation
    • Helical secondary structure in target conformation
    • DSSP secondary structure prediction in target conformation
    • Number of clashes in target conformation
    • Chain in alternative conformation
    • Amino acid in alternative conformation
    • Phi angle in alternative conformation
    • Psi angle in alternative conformation
    • Trans proline angle in alternative conformation
    • Helical secondary structure in alternative conformation
    • DSSP secondary structure prediction in alternative conformation
    • Number of clashes in alternative conformation
    • Compatible with target conformation
    • Compatible with alternative conformation
    • Compatible with target and not with alternative conformation
  • Option 2 (with target conformation input only) columns include:
    • Chain in target conformation
    • Residue number in target conformation
    • Amino acid in target conformation
    • Phi angle in target conformation
    • Psi angle in target conformation
    • Trans proline angle in target conformation
    • Helical secondary structure in target conformation
    • DSSP secondary structure prediction in target conformation
    • Number of clashes in target conformation
    • Compatible with target conformation
  • Option 3 (with alternative conformation input only) columns include:
    • Chain in alternative conformation
    • Residue number in alternative conformation
    • Amino acid in alternative conformation
    • Phi angle in alternative conformation
    • Psi angle in alternative conformation
    • Trans proline angle in alternative conformation
    • Helical secondary structure in alternative conformation
    • DSSP secondary structure prediction in alternative conformation
    • Number of clashes in alternative conformation
    • Compatible with alternative conformation

Example/Test

RSV F example

Copy all required files into a directory (Files: CRISPro_v.1.1-2.R, rama8000-transpro.data, mutate.py, 4jhw_trimer.pdb, and 3rrr_trimer.pdb)

• Option 1: R --vanilla < CRISPro_v.1.1-6.R 1.5 0.01 8 rama8000-transpro.data mutate.py RSV_F_option-1 1 4jhw_trimer.pdb A 3rrr_trimer.pdb A
• Option 2: R --vanilla < CRISPro_v.1.1-6.R 1.5 0.01 8 rama8000-transpro.data mutate.py RSV_F_option-2 2 4jhw_trimer.pdb A
• Option 3: R --vanilla < CRISPro_v.1.1-6.R 1.5 0.01 8 rama8000-transpro.data mutate.py RSV_F_option-3 3 3rrr_trimer.pdb A

Contact

Reda Rawi: reda.rawi@nih.gov