MemPrO

Membrane Protein Orientation in Lipid Bilayers. The paper associated with this code can be found here.

Code and parameters from the following were used to help write this code:

Insane: Tsjerk A. Wassenaar et al. "Computational lipidomics with insane: A versatile tool for generating custom membranes for molecular simulations". In: Journal of Chemical Theory and Computation 11 (5 May 2015), pp. 2144–2155. issn: 15499626. GitHub and paper

Martini forcefield: Siewert J. Marrink et al. "The MARTINI Force Field: Coarse Grained Model for Biomolecular Simulations". In: (2007). Paper

Peptidoglycan parameters: Rakesh Vaiwala et al. "Developing a Coarse-Grained Model for Bacterial Cell Walls: Evaluating Mechanical Properties and Free Energy Barriers". In: Journal of Chemical Theory and Computation 16 (8 Aug. 2020), pp. 5369–5384. issn: 15499626. Paper

Installation

pip (recommended)

pip install mempro

From source

Clone the GitHub repository. Python 3.11 or later is required, along with the following packages:

• JAX 0.6.0 (CPU). Note: only JAX 0.6.0 is supported — versions 0.6.1 and later introduced breaking changes to the CPU runtime that cause crashes or significant performance regressions:

pip install "jax[cpu]==0.6.0"

• Matplotlib 3.8.4 or later:

pip install "matplotlib>=3.8.4"

A Martini 3 forcefield file is also required and can be downloaded from here. When running from source, place the .itp file in the same directory as MemPrO.py, or supply its path using the -itp flag.

MemPrO is also accessible on Google Colab via the link [ADD LINK]. The Colab will contain instructions for use. (Currently the Colab is WIP and this will be updated.)

Running MemPrO

From source

python PATH/TO/MemPrO.py -f input_file.pdb

Installed via pip

mempro -f input_file.pdb

No environment variables are required. By default MemPrO will use all available CPU cores and look for Insane4MemPrO.py and martini_v3.itp in the same directory as the script (or the installed package directory). These paths can be overridden with the -insane and -itp flags if needed.

Outputs

MemPrO is intended to be run on PDB files, and will output the following files:

• orientation.txt — Contains general information about all of the final minima. In general rank 1 is the best orientation, however there are cases where this is not true. orientation.txt can then be used to determine the best orientation.

• local_minima_orientation.png — A representation of how spread out the local minima are. The more white the image, the more confidence can be placed in rank 1. This is only true in general and there are some cases where this can be safely ignored.

• local_minima_potential.png — A representation of the potential of the final configuration from a given starting configuration. This complements local_minima_orientation.png but in general does not provide much additional information.

• Rank_{n}/ — A folder containing all relevant information for a particular rank:

  • oriented_rank_{n}.pdb — The nth ranked configuration. Contains the protein (atomistic or coarse grain depending on the input) and a dummy membrane. The rank is based on number of hits (or potential, see -rank flag).
  • info_rank_{n}.txt — Some information about the orientation.
  • CG_System_rank_{n}/ — A CG system built using Insane4MemPrO for the rank N configuration, including a topology file. Only produced when using -bd and -bd_args.
  • PG_potential_curve.png — The potential curve for the position of the PG cell wall. Only produced with -pg.
  • cross-section_area_curve.png — The cross-sectional area of the segment of the protein passing through the PG layer. Only produced with -pg.
  • Z_potential_curve.png — Potential against Z position of the final orientation. Gives an idea of the nature of the minima.
  • curv_potential_curve.png — Potential against global membrane curvature for the final orientation. Can indicate whether curvature minimisation should be used.

Flags

-h, --help — Displays a help message with all possible flags.

-f, --file_name — Input PDB file for the protein to orient. The protein can be either atomistic or coarse grained; the code detects which automatically and ignores unknown atom/bead types. It is recommended that the PDB file has no missing atoms, as this can reduce orientation quality.

-o, --output — Name of the output directory. Defaults to Orient/ in the current working directory.

-ni, --iters — Maximum number of minimisation iterations. The default of 150 is sufficient in most cases. Reducing this value increases speed; increasing it can sometimes improve results on difficult systems.

-ng, --grid_size — Number of initial starting configurations. Defaults to 36. For best efficiency this should be a multiple of the number of CPUs being used. More starting configurations sample the space of minima better, but there are diminishing returns and generally no more than 40 are needed.

-nc, --num_cpus — Number of CPU cores to use. Defaults to all available cores. For best performance with -ng, set -nc to a value that divides -ng evenly.

-rank, --rank — Ranking method for minima: h ranks by percentage hits, p ranks by potential, auto (default) ranks by a value calculated from approximate minima depth and potential.

-dm, --dual_membrane — Toggles dual membrane minimisation. Splits the membrane into an inner and outer membrane and minimises the distance between them. Only use this if your protein spans the periplasm or is a gap junction etc.

-ch, --charge — Partial charge of the (inner) membrane. The value corresponds to the average charge on a lipid divided by the average area per lipid, representing the average charge across the bilayer sheet. For an E. coli inner membrane this is around -0.008. Default is 0, which gives the best performance in most cases.

-ch_o, --charge_outer — Charge for the outer membrane only. Has no effect without -dm.

-mt, --membrane_thickness — Initial thickness of the (inner) membrane in Angstroms. Default is 28.

-mt_o, --outer_membrane_thickness — Initial thickness of the outer membrane in Angstroms. Has no effect without -dm. Default is 24.

-mt_opt, --membrane_thickness_optimisation — Toggles membrane thickness optimisation. Cannot be used with -c.

-tm, --transmembrane_residues — Indicates which residues are expected to be transmembrane. Format is a comma-separated list of inclusive-exclusive ranges e.g. 10-40,50-60. Intended only for situations where the transmembrane region is known and MemPrO is consistently orienting incorrectly. Cannot be used with -w.

-pg, --predict_pg_layer — Toggles PG cell wall prediction for dual membrane minimisations. Outputs a dummy PG layer at the lowest potential position and two diagnostic graphs. Due to the nature of the PG layer the lowest potential is not necessarily the biologically correct placement; it is recommended to use the graphs for further interpretation. Requires -dm.

-pg_guess, --pg_layer_guess — An initial guess for the PG layer position, typically based on standard PG layer positions in a specific bacterium.

-pr, --peripheral — Use an alternative method for determining initial insertion depth. The usual method places the weighted mean of the hydrophobic residues at the membrane centre, which does not work well for peripheral membrane proteins. The alternative method scans all possible insertion depths within a range. Does not work with -dm.

-w, --use_weights — Toggles use of b-factors to weight the minimisation. Useful if part of your protein is particularly flexible or poorly predicted by AlphaFold. Do not use this if all b-factors are 0. Cannot be used with -tm.

-wb, --write_bfactors — Writes individual bead potentials to the b-factors of the output PDB. Useful for diagnosing orientation problems. Not currently compatible with -c.

-c, --curvature — Toggles global curvature minimisation. Not currently compatible with -dm or -mt_opt.

-flip, --flip — Flips the protein in the Z-axis on output.

-itp, --itp_file — Path to a Martini 3 .itp file. Defaults to martini_v3.itp in the same directory as MemPrO.py (or the installed package directory).

-insane, --insane_path — Path to Insane4MemPrO.py. Defaults to the same directory as MemPrO.py (or the installed package directory). Only required when using -bd.

-bd, --build_system — Number of final ranked configurations to build as CG MD-ready systems using Insane4MemPrO. Currently only works with coarse grain input files.

-bd_args, --build_arguments — Arguments to pass to Insane4MemPrO, primarily lipid composition, solvent type etc. Required when using -bd.

-res, --additional_residues — Comma-separated list of additional residues present in the input file (e.g. POPE,POPG).

-res_itp, --additional_residues_itp_file — An .itp file describing each additional residue. Required when using -res.

-res_cg, --residue_cg_file — A folder containing files named RES.pdb for each additional residue, providing coarse graining information. Examples can be found in the CG2AT repository. Only required for atomistic inputs with additional residues.

Examples

The examples below use python PATH/TO/MemPrO.py; if installed via pip, replace this with mempro.

Orient input_file.pdb on a grid of 40 starting configurations with 150 minimisation iterations:

python PATH/TO/MemPrO.py -f input_file.pdb -o Output_dir/ -ng 40 -ni 150

Orient with global curvature minimisation and write per-bead potentials to b-factors:

python PATH/TO/MemPrO.py -f input_file.pdb -o Output_dir/ -ng 40 -ni 150 -c -wb

Orient with a dual membrane system, then build the top 2 ranked configurations as CG systems. The inner membrane contains POPE, POPG and CARD; the outer membrane contains LIPA:

python PATH/TO/MemPrO.py -f input_file.pdb -o Output_dir/ -ng 40 -ni 150 -dm -bd 2 -bd_args "-negi_c0 CL -posi_c0 NA -sol W -l POPE:7 -l POPG:2 -l CARD:1 -uo LIPA"

Orient using 8 CPU cores:

python PATH/TO/MemPrO.py -f input_file.pdb -o Output_dir/ -nc 8

Orient using a GPU:

python PATH/TO/MemPrO.py -f input_file.pdb -o Output_dir/ -p gpu

A more detailed set of tutorials is available here.

Insane4MemPrO

MemPrO comes with Insane4MemPrO, a CG system builder based on Insane. Insane4MemPrO allows the user to build more complex systems with up to 2 membranes, curvature, multiple proteins and more. When used with MemPrO directly via the -bd flag the system is built automatically.

Flags

I/O related flags

-f — (Optional) Input protein to build a CG system around.

-o — (Required) Output file name.

-p — (Optional) Output topology file.

-ct — (Optional) Creates a template file. B-factors can be edited to indicate placement of multiple proteins. See examples below.

-in_t — (Optional) Input a template created by -ct. Builds a CG system with proteins (supplied via -fs) placed at the positions indicated in the template.

-fs — (Optional) A text file with an ordered list of proteins for multiple protein placements.

System size

-x — (Required) Box size in the x dimension.

-y — (Required) Box size in the y dimension.

-z — (Required) Box size in the z dimension.

Membrane/lipid options

-l — (Optional) Lipid type and relative abundance (NAME[:N]) in the membrane (or lower leaflet only if -u is used).

-u — (Optional) Lipid type and relative abundance (NAME[:N]) in the upper leaflet.

-lo — (Optional) Lipid type and relative abundance (NAME[:N]) in the outer membrane (or lower leaflet if -uo is used).

-uo — (Optional) Lipid type and relative abundance (NAME[:N]) in the outer membrane upper leaflet.

-a — (Default: 0.6) Area per lipid (nm²) in the membrane (or lower leaflet if -au is used).

-au — (Optional) Area per lipid (nm²) in the upper leaflet.

-ao — (Optional) Area per lipid (nm²) in the outer membrane (or lower leaflet if -auo is used).

-auo — (Optional) Area per lipid (nm²) in the outer membrane upper leaflet.

-ps — (Default: 0) Distance between inner and outer membrane.

-curv — (Default: 0,0,1) Membrane curvature: three comma-separated values for curvature at the peak, curvature at the base, and direction of curvature.

-curv_o — (Default: 0,0,1) Curvature of the outer membrane. See -curv.

-curv_ext — (Default: 3) Extent of the curved region in the absence of a protein.

-micelle — (Optional) Builds a micelle around the protein instead of a bilayer.

-radius — (Optional) Builds a membrane disk with the given radius, useful for simulating nanodiscs.

-def — (Optional) Takes as input the Membrane_data folder generated by MemPrOD. Builds the deformations predicted by MemPrOD. Cannot be used with -curv or -ps.

Peptidoglycan layer options

-pgl — (Optional) Number of PG layers to place at -pgl_z.

-pgl_z — (Optional) Z position of the PG layer relative to the centre of the periplasmic space.

-cper — (Optional) Percentage of cross-links.

-lper — (Optional) Percentage of cross-links that are between layers.

-per33 — (Optional) Percentage of 3-3 cross-links; all other cross-links will be 3-4.

-oper — (Optional) Percentage chance of a monomer linking with an oligomer. (Actual chance of link is cper * oper.)

-gdist — (Default: 0.75,4,8.9,0.25,10,45) Distribution of glycan strand lengths. Format as weight1,stddev1,mean1,weight2,... where each triple describes a Gaussian. The sum of these Gaussians forms the distribution.

Protein options

-fudge — (Default: 0.3) Fudge factor for allowing lipid-protein overlap.

Solvent options

-sol — (Required) Solvent type.

-sold — (Default: 0.5) Solvent packing density.

-solr — (Default: 0.1) Magnitude of random deviations to solvent positions.

Charge options

-posi_c0 — (Required) Positive ion type and relative abundance (NAME[:N]) in the system (or compartment 0 if -posi_c1/-posi_c2 are also used). When using multiple membranes, disjoint water compartments may form; these flags allow each compartment to be neutralised independently with different ions.

-negi_c0 — (Required) Negative ion type and relative abundance (NAME[:N]) in the system (or compartment 0).

-posi_c1 — (Optional) Positive ion type and relative abundance in compartment 1.

-negi_c1 — (Optional) Negative ion type and relative abundance in compartment 1.

-posi_c2 — (Optional) Positive ion type and relative abundance in compartment 2.

-negi_c2 — (Optional) Negative ion type and relative abundance in compartment 2.

-ion_conc — (Default: 0.15,0.15,0.15) Ion concentration in each compartment.

-charge — (Default: auto) Charge of the system. auto detects charge automatically.

-charge_ratio — (Optional) How to split the charge across compartments. If not supplied, each compartment is neutralised separately.

-zpbc — (Optional) Determines whether Z periodicity is used when calculating compartments.

Examples

These instructions walk through building a CG system with a curved membrane containing multiple proteins.

First, create a template:

python PATH/TO/Insane4MemPrO.py -l POPE -sol W -x 10 -y 10 -z 50 -o test.gro -p topol.top -curv 0.1,0.15,1 -fudge 0.3 -curv_ext 6 -ct template.pdb -negi_c0 CL -posi_c0 NA

This creates a POPE-only membrane in a (10,10,50) box (automatically enlarged if needed to accommodate the curvature). The curvature 0.1,0.15,1 specifies a peak curvature of 0.1, a base curvature of 0.15, and the curved region pointing in the positive Z direction, maintained for 6 Å before returning to planar.

Next, open template.pdb in PyMOL. Protein positions are indicated by b-factors. Select a single bead at each desired protein location and run alter sele,b=N where N increments by 1 for each protein (the Nth protein will be placed at the bead with b-factor N). Once all b-factors are set, run save template.pdb.

Then build the final system:

python PATH/TO/Insane4MemPrO.py -l POPE -sol W -x 10 -y 10 -z 50 -o test.gro -p topol.top -curv 0.1,0.15,1 -fudge 0.3 -curv_ext 6 -in_t template.pdb -fs prots.txt -negi_c0 CL -posi_c0 NA

Ensure all membrane-relevant values match the template command. -in_t supplies the edited template and -fs supplies a text file listing the same number of proteins as edited b-factors. The -p flag produces a rudimentary .top file; protein-related entries will need to be updated but all other values are correct.

More detailed tutorials are available here.

My protein didn't orient correctly

If your protein hasn't oriented correctly there are a number of things to try:

• Check all orientations by loading orientations.pdb and reviewing orientations.txt. The correct orientation may not be rank 1.
• Check curv_potential_curve.png to see if your protein prefers a curved membrane. If so, using -c will help.
• Check the PDB file for missing atoms or residues, which can cause the surface to be incorrectly evaluated. Missing atoms should be fixed before orientation.
• Using -wb can be helpful for diagnosing missing atoms — it writes the potential contribution of each residue as a b-factor, viewable in PyMOL.
• If your protein is a peripheral membrane protein, using -pr will help greatly. In some cases -pr will also help integral membrane proteins.
• MemPrO runs with a membrane charge of 0 by default. Some proteins that associate with the membrane through charge interactions will not orient correctly at 0 charge. For these, using -ch (or -ch_o for the outer membrane) to set the charge will help. Values around -0.005 are a good starting point.

FAQ

There are currently no frequently asked questions. If you have any questions or encounter errors you cannot fix, please contact phillip.stansfeld@warwick.ac.uk.