The Outer Membranes of Gram-negative bacteria are asymmetric lipid bilayers, composed by standard lipids in the inner leaflet and mostly lipopolysaccharides (LPSs) in their outer leaflet. Outer membranes and their components are of critical importance for bacterial cell structure and function, as outer membrane proteins can regulate a diverse range of functions, while the organization and dynamics of LPSs can influence bacterial resistance against antimicrobial agents and cause toxic reactions in human hosts, leading to a number of diseases.
Despite their significance, the experimental study of outer membranes is challenging. Molecular Dynamics (MD) simulations can be a useful tool in modeling the structure and dynamics of membranes and membrane proteins. However, the preparation and simulation of outer membrane proteins in their native LPS-containing outer membrane environment is not straightforward.
The Gram-Negative Outer Membrane Modeler (GNOMM) is an automated workflow for the construction of LPS-rich outer membrane systems in MD simulations. GNOMM currently supports four widely used force fields, namely, the CHARMM36 all-atom, GROMOS 54A7 united-atom, MARTINI coarse-grained and PACE hybrid resolution models, and enables the building of membrane and protein-membrane systems with complex lipid bilayers containing LPSs. The generated output configurations can be subsequently used to perform Molecular Dynamics simulations with the freely available, high performance GROMACS simulation engine.
GNOMM is publicly available through http://bioinformatics.biol.uoa.gr/GNOMM.
Users can start using GNOMM through the Submit page. The submission form is presented in Fig. 1:
Figure 1.The Submission Form of GNOMM
GNOMM currently supports the creation of three different types of simulation systems:
To build a "Protein/Membrane" system, select the option "Protein/Membrane" in the Submit page (Fig. 2).
To proceed with the submission, you can either enter a valid PDB ID or upload a structure file in PDB format (note that, for efficiency, the maximum size of PDB uploads is currently 30 MBs). Note that the structure should be pre-oriented with respect to the membrane plane. This means that the PDB's Z- principal axis needs to be aligned to the membrane plane's normal. Such files can be retrieved from the Orientations of Proteins in the Membrane (OPM) database or the Protein Data Bank of Transmembrane Proteins (PDB-TM), or can be generated through the PPM server. Using RCSB PDB as the source of input, although available, is generally NOT recommended since the structures are not likely to have the proper orientation.
Figure 2.Example submission of a "Protein/Membrane" system for OmpA (PDB: 1BXW), retrieved from OPM.
If no errors occur, you will be redirected to the system setup page. Otherwise, an error message will appear and you will be prompted to go back and change your submission.
The system setup page is divided into three sub-section, corresponding to the three main steps followed by the GNOMM modeling process:
Figure 3. The protein preparation form
Figure 4. Definition of membrane dimensions
Use the form, as shown in Fig. 4, to enter the X and Y values of the bilayer's dimensions. Make sure that these are large enough to include your protein and leave ample space for the packing of lipids, and to properly respect periodic boundary conditions. To aid you, GNOMM calculates the unit cell dimensions of your submitted protein and, based on those, provides the minimum suggested values for you to enter.
Note that X and Y values may not necessarily be the same. You can enter different values and produce non-square membrane patches if you need.
Also note that for computational efficiency, the membrane surface area must not exceed 90000 Å2.
After you have defined the membrane's dimensions, you need to define its composition, by filling out the table shown in Fig. 5:
Figure 5. Definition of membrane composition
Fill the table with the percentage ratios you want in your table. Change only the values of the lipids you actually want to use, leave the rest at zero (0). Make sure that the total sum of each leaflet is 100% (100).
In the figure's example we want a bilayer with a uniform Ra LPS (RAMP) upper leaflet and a uniform POPE lower leaflet. Therefore, we enter 100 for RAMP in the upper leaflet and 100 for POPE in the lower leaflet.
In step 3 (Fig. 6) you choose whether you want to add solvent layers on each side of your system and whether you want to add ions:
Figure 6. Solvation and ionization
After your job is submitted, you will be redirected to the following page (Fig. 7):
Figure 7. The job process page
Since building may take a while, you are strongly advised to bookmark this page and return later. Alternatively, you can use your job ID to check your status through the "Retrieve Results" form (see the relevant section below).
When your job is complete, you will see the following page (Fig. 8):
Figure 8. The results page
Click the provided link to download a compressed file with all your results (Fig. 9). These will include the following:
Figure 9. Contents of the results
You can retrieve the results of your previous jobs or check the progress of a currently running job by using the form in the "Retrieve Results" page. In the form, just input the ID corresponding to the job you submitted.
Figure 10. The Retrieve Results page
To build a "Membrane-only" system, simply select the option "Membrane-only" in the Submit page, choose a force field and click the "Submit" button (Fig. 11):
Figure 11. The "Membrane-only" option in the Submit page.
Since you are building a Membrane-only system, the first part of the GNOMM modeling process (protein manipulation) is skipped. The rest of the process (membrane building, solvation and ionization) is *exactly* the same as in the Protein-Membrane builder interface.
To build a system using a pre-processed PDB structure and its acompanying topology, select the "Upload pre-processed PDB and topology (for advanced users)" option in the Submit page (Fig. 12):
Figure 12. Example submission of a system with a pre-processed PDB structure and topology.
In the example of Fig. 12, we prepare a simulation for OmpF in complex with ampicillin (AIC), an antibiotic (PDB: 4GCP). The protein has been pre-processed for use with GROMACS using pdb2gmx and the CHARMM36 force field. For AIC, force field parameters have been generated using CGenFF.
Note that the process of building a protein-ligand system for simulations requires an intermediate (at least) knowledge of how GROMACS works. There is an excellent tutorial in Prof. Justin Lemkul's GROMACS Tutorial site (www.mdtutorials.com/gmx/) for the construction of protein-ligand systems (see here). You are advised to consult this tutorial and any other relevant material to familiarize yourself with the subject.
Fotis A. Baltoumas, Stavros J. Hamodrakas & Vassiliki A. Iconomidou (2019)
The Gram-Negative Outer Membrane Modeler: automated building of lipopolysaccharide-rich bacterial outer membranes in four force fields
J. Comput. Chem. Jul 5; 40(18): 1727-1734, DOI: 10.1002/jcc.25823