Building and Analyzing a Complex Lipid Bilayer and Embedding a Membrane Protein

Tutorial Created with Software Release: 2025-4
Topics: Biologics Drug Discovery, Consumer Packaged Goods, Small Molecule Drug Discovery
Methodology: All-Atom Molecular Dynamics
Products Used: BioLuminate, Desmond, MS Complex Bilayer Builder, MS Maestro, Maestro

Tutorial files

2.3 GB
This tutorial is written for use with a 3-button mouse with a scroll wheel.
Words found in the Glossary of Terms are shown like this: Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

 

Tip: You can hover over a glossary term to display its definition. You can click on an image to expand it in the page.
Abstract:

 

In this tutorial, we will learn how to create a multi-component lipid bilayer and how to embed a membrane protein into one using the Complex Bilayer Builder panel. We will then use the Membrane Analysis panels to analyze the typical biophysical properties of a bilayer, such as area per lipid and bilayer thickness.

 

Tutorial Content

  1. Introduction to Complex Lipid Bilayers and Membrane Proteins

  1. Creating Projects and Importing Structures

  1. Building and Analyzing a Multi-Component Lipid Bilayer

  1. Building a Multi-Component Lipid Bilayer Including a Membrane Protein

  1. Conclusion and References

  1. Glossary of Terms

1. Introduction to Complex Lipid Bilayers and Membrane Proteins

Lipid bilayers and membrane proteins are important targets in drug and active ingredient discovery, in drug delivery and formulation of skin related personal care products. Properties of membrane proteins often are largely related to the exact lipid composition of the relevant membrane. This makes simulation of complex lipid bilayer systems on an atomic level highly desirable.

The Complex Bilayer Builder panel allows for easy creation of all-atom models of single or multi-component lipid membranes through a multistage build and relaxation procedure. In a first step, the system is assembled by packing the appropriate number of lipid molecules in a defined ratio into the simulation box. This is followed by a relaxation protocol consisting of several stages of short and restrained molecular dynamics (MD) simulations amounting to under 1 ns, where the restraints are slowly released from stage to stage. This is to enhance the structure and reduce any packing defects, but is not an equilibration of the system. Only after sufficient further equilibration and a production run (e.g. with the MD Multistage Workflow or the Molecular Dynamics panel), the Membrane Analysis panels can then calculate and plot the area per lipid (APL), bilayer thickness, and other standard properties over the course of an MD trajectory.

The structural information of a membrane protein can be specified to include its position in the bilayer based on experimental data. The Complex Bilayer Builder panel will notify you if the protein is already appropriately positioned in a bilayer when OPM (Orientations of Proteins in Membranes) data is found. Otherwise, you can manually position the protein in the coordinate space.

In this tutorial, we will use the Complex Bilayer Builder panel to construct and relax a POPC-POPE membrane system (demonstrated in Section 3) and visually analyze the generated system. Subsequently, we will perform a molecular dynamics (MD) simulation with the MD Multistage Workflow panel and analyze the trajectory with the Membrane Analysis and the Membrane Analysis Viewer panels, to calculate the area per lipid and the membrane thickness, two common descriptors in membrane biophysics.

Finally, we will use the Complex Bilayer Builder panel to construct a lipid bilayer containing an embedded membrane protein (demonstrated in Section 4), the transmembrane membrane protein beta2 adrenoceptor - a G-protein-coupled receptor (GPCR).

2. Creating Projects and Importing Structures

At the start of the session, change the file path to your chosen Working Directorythe location where files are saved in MS Maestro to make file navigation easier. Each session in MS Maestro begins with a default Scratch Projecta temporary project in which work is not saved, closing a scratch project removes all current work and begins a new scratch project, which is not saved. A MS Maestro project stores all your data and has a .prj extension. A project may contain numerous entries corresponding to imported structures, as well as the output of modeling-related tasks. Once a project is saved, the project is automatically saved each time a change is made.

Structures can be built in MS Maestro or can be imported using File > Import Structures (or drag-and-dropped), and are added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and Project Tabledisplays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data. The Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion is located to the left of the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed. The Project Tabledisplays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data can be accessed by Ctrl+T (Cmd+T) or Window > Project Table if you would like to see an expanded view of your project data.

OROR

  1. Double-click the Materials Science, Maestro or BioLuminate icon

Note: This workflow can be performed in MS Maestro, Maestro or BioLuminate. Use whichever interface you are comfortable with or typically use for your projects. This tutorial uses MS Maestro.

Figure 2-1. Change Working Directory option.

  1. Go to File > Change Working Directory
  2. Find your directory, and click Choose
  3. Pre-generated files are included for running jobs or examining output. Download the zip file here: https://www.schrodinger.com/sites/default/files/s3/release/current/Tutorials/zip/complex_bilayer.zip
  4. After downloading the zip file, unzip the contents in your Working Directorythe location where files are saved for ease of access throughout the tutorial

Figure 2-2. Save Project panel.

  1. Go to File > Save Project As
  2. Change the File name to complex_bilayer_tutorial, click Save
    • The project is now named complex_bilayer_tutorial.prj

3. Building and Analyzing a Multi-Component Lipid Bilayer

In this section, we will build a two-component lipid bilayer containing the two phospholipids POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) surrounded by water.

After the assembly and a short automated relaxation with the Complex Bilayer Builder panel, we will visually inspect the system. We will then run an MD simulation with the MD Multistage Workflow panel and calculate bilayer properties such as the area per lipid and thickness from the MD trajectory to analyze the generated system. This will be done with the Membrane Analysis and Membrane Analysis Viewer panels.

3.1 Building a two-component lipid bilayer

Figure 3-1. The Complex Bilayer Builder panel.

  1. Go to Tasks > Materials > Structure Builders > Complex Bilayer Builder

The first section of the panel (Membrane Protein) is for specifying and positioning a membrane protein in the bilayer. We will use it in the second part of the tutorial (Section 4).

The Lipid Bilayer section contains all options for the dimensions and components of the lipid bilayer.

In the Solvents and Ions section, options for the water solvent and ions for both neutralization and ionic strength are available.

Finally, the last section offers options for the MD relaxation after the assembly of the system. It can be used to append custom MD relaxation steps via an MSJ file, which are then run after the default relaxation protocol.

Figure 3-2. Setting up the dimensions and composition of the lipid bilayer.

  1. In the Lipid Bilayer section, set the Box dimensions to 100 Å in X and Y direction

The default of 50 by 50 Å is the minimal system size.

  1. Make sure the option for Symmetric composition across leaflets is checked
    • This keeps both the upper and lower leaflet identical
  2. Below the lipid table, click Add Lipid…
    • A new entry in the lipid table is created
  3. Click on the newly created POPC entry to open the dropdown menu and change the lipid type to POPE
  4. Ensure that the bilayer composition is a 50:50 ratio of POPC to POPE

The optimal size of your system depends on the application and type of system under study. In this tutorial, we are interested in macroscopic properties, such as the average area per lipid and membrane thickness, so we opt for a system larger than the default setting. If you were to investigate a small molecule interaction with a membrane, you could work with a smaller system, always remembering to account for sufficient distance between the PBC images. Please note that your system may shrink during equilibration. For this reason, we recommend building a slightly larger system than your desired final size.

Rather than creating a symmetrical composition of both leaflets, you can also construct the upper and lower leaflets of the bilayer with an asymmetric composition, featuring differences in the types and ratios of lipids. This is advanced model building and requires extra consideration of the biophysical aspects of asymmetric bilayers.

Available phospholipids are POPC, POPE, DPPC and DMPC. In addition, cholesterol (CHL) is available. Besides the provided components, you can import your own custom lipids either via SMILES or a molecular structure from the workspace.

Figure 3-3. Specifying solvent and ions for the environment of the lipid bilayer and running the job.

  1. Make sure the Water Padding is set to 12 Å
    • This defines the height of water to add above and below the membrane
  2. Change the Job Name to complex_bilayer_POPC-POPE
  3. Adjust the job settings as needed
    • This job requires a GPU host for the MD relaxation steps after the system is assembled. It can be completed in about 45 minutes.
  4. If you would like to run the job yourself, click Run. Otherwise, import the provided results from the zip file via File > Import Structures: Section_03 > complex_bilayer_POPC-POPE > complex_bilayer_POPC-POPE-out.cms
  5. Close the Complex Bilayer Builder panel

Besides the SPC (default) and SPCE water models, several TIP water models are available. For information regarding the FF parametrization, see the OPLS Force Field documentation.

Ensure the water padding is sufficient so that self-interactions in the Z dimension are prevented. Approx 1 nm of space between lipid and box boundaries on both sides should be sufficient.

Our system only consists of zwitterionic (net-neutral) lipids and water, so no ions are needed to neutralize the system. Neutralization is relevant if your system contains charged lipids. The types of ions for neutralizing the system and an additional salt concentration (e.g. to mimic physiological or experimental conditions) can be specified.

3.2 Visually analyzing the two-component lipid bilayer

Figure 3-4. Selecting the hydrophilic atoms of the lipids.

Now we will visually inspect the assembled and relaxed lipid bilayer.

 

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue and select(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries the Assembled system entry
  2. In the top toolbar click Define…
    • The Atom Selection panel opens
  3. In the Atom tab, scroll down to and select hydrophilic, then check the option for true, and click Add
    • The box at the bottom showing the atom selection language (ASL) should be updated
    • This selects all atoms in the bilayer labeled as hydrophilic
    • It is also possible to select the hydrophobic atoms
  4. Now click OK
  5. Display the selected atoms in the CPK representation
    • You can see the hydrophilic atoms of the head groups, oxygen in red and nitrogen in blue

Figure 3-5. Visually inspecting the assembled and relaxed lipid system.

  1. In the Quick Select section in the top toolbar, click All to select all atoms in the system
  2. In the Style dropdown menu, click show polar hydrogens only
  3. In the Quick Select section, click Water
  4. In the Style dropdown menu, choose the  ball-and-stick representation

Now you can visually inspect the system.

In a correctly assembled and relaxed lipid bilayer, the lipid headgroups should not be present near the center of the bilayer and the tails should point towards the inside. Any packing defects in the bilayer system after the initial relaxation can usually be resolved by additional equilibration via MD simulations, but this should be an unlikely event.

Additionally, there should not be a large number of water molecules forming a slab within the bilayer or a pore through the bilayer. If a few individual water molecules are found within the membrane, this can usually be resolved through further MD equilibration.

In this system, there are no lipid head groups near the center of the bilayer and no lipid tails are sticking out. Additionally, no water molecules reside inside the bilayer. The system is thus properly assembled and ready to proceed for MD simulations.

3.3 Running an MD Simulation

In the following, we will run an MD simulation for equilibration via the MD Multistage Workflow panel. In case you do not have access to the Materials Science suite, but are following along in either Maestro or BioLuminate, you can either directly import the provided results or use the Molecular Dynamics panel to run an MD simulation. You can find more information on the panel usage in the Introduction to All-Atom Molecular Dynamics Simulations with Desmond tutorial. It is also possible to supply an MSJ file via the custom MSJ option in the Complex Bilayer Builder panel to run MD equilibration steps after the assembly and initial relaxation of the system directly within the builder panel.

Figure 3-6. The MD Multistage Workflow panel.

  1. Use the WAM (workflow action menu) button () to open the MD Multistage Workflow panel
    • The MD Multistage Workflow panel opens
    • Alternatively, access the panel via Tasks > Materials > Classical Mechanics > MD Simulations > MD Multistage Workflow

Figure 3-7. Setting up the MD job.

  1. Select the option to Remove center of mass motion
  2. In the already existing stage (Brownian Minimization), change the Stage type to Molecular Dynamics
  3. Change the Simulation time to 250 ns
  4. Change the Ensemble class to NPγT and check that the Surface tension is set to 0
  5. Change the Job Name to multistage_simulation_POPC-POPE_250ns
  6. Adjust the job settings as needed
    • This job requires a GPU host and should take about 1.5 days to complete
  7. If you would like to run the job yourself, click Run. Otherwise, import the provided results from the zip file via File > Import Structures: Section_03 > multistage_simulation_POPC-POPE_250ns > complex_bilayer_POPC-POPE_250ns-out.cms
  8. Rename the new entry group to MD Multistage Simulation
  9. Close the MD Multistage Workflow panel

When working with systems containing a lipid bilayer, we recommend to run in the NPγT ensemble with zero surface tension. Another option could be the thermodynamically equivalent NPT ensemble with semi-isotropic pressure coupling, but it might take longer to equilibrate.

Further information on how to run MD simulations with the MD Multistage Workflow panel can be found in the Disordered System Building and Molecular Dynamics Multistage Workflows tutorial.

3.4 Calculating area per lipid and bilayer thickness

Figure 3-8. Setting up the Membrane Analysis job.

Now we will run a short analysis calculation to estimate the area per lipid and thickness of the bilayer.

  1. Ensure the new entry is selected(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries and includedthe entry is represented in the Workspace, the circle in the In column is blue

Note: Feel free to visualize the hydrophilic head groups and water molecules along the trajectory as in the beginning of this section. In this case load the trajectory first by clicking on the T button () next to the entry and selecting Load Trajectory.

  1. Go to Tasks > Materials > Classical Mechanics > Trajectory Analysis > Membrane Analysis Calculations
  2. Click Load from Workspace
    • The system is loaded into the panel, the number of frames are shown
    • The trajectory frames for the analysis calculation can be chosen, the default is the full trajectory
  3. Ensure the option for Thickness and area per lipid is selected
    • The number of selected atoms and the ASL should update
    • You can highlight the hydrophilic atoms in the workspace
  4. Uncheck the CH bond order parameter
  5. Change the Job name to membrane_analysis_POPC-POPE_250ns
  6. Adjust the job settings as needed
    • This job requires a GPU subhost for the analysis of the MD simulation. It can be completed in about 5 minutes.
  7. If you would like to run the job yourself click Run, otherwise import the provided results from the zip file via File > Import Structures: Section_03 > membrane_analysis_POPC-POPE_250ns > membrane_analysis_POPC-POPE_250ns-out.maegz
  8. Rename the new entry group to Bilayer Analysis
  9. Close the Membrane Analysis panel

Figure 3-9. The Summary tab in the Membrane Analysis Viewer.

  1. Use the WAM (workflow action menu) button () to open the Membrane Analysis Viewer panel
    • Alternatively, access the panel via Tasks > Materials > Classical Mechanics > Trajectory Analysis > Membrane Analysis Results
  2. Click Load from workspace to import the results
  3. Have a look at the system information in the Summary tab
  4. Then, switch to the Area per Lipid tab

The Summary tab shows the composition of both leaflets for the analyzed bilayer, including the number of atoms, molecules, their weight and total charge. You can already see the average area per lipid and average membrane thickness for the second half of the simulation. You can visualize both properties versus the simulation time in the following tabs.

In this system, a single POPE molecule was removed from the lower leaflet during the packing in the assembly step by the Complex Bilayer Builder panel due to bad placement. The difference of a single lipid has no effect on the overall composition or behavior of the bilayer, but a large mismatch in a symmetric bilayer should be investigated further.

Figure 3-10. The Area per Lipid tab in the Membrane Analysis Viewer.

  1. Change the Block size for averaging to 10 ns
  2. You can shift the two dashed blue sliders via drag-and-drop to find a converged area per lipid, the value at the bottom is updated automatically

The area per lipid (APL) is the area of the membrane plane occupied by a single lipid molecule within the bilayer, and it is a parameter that is specific to membrane structure and composition. An APL value that fluctuates around a stable mean value can be used to judge the convergence of a simulation, indicating sufficient equilibration of the bilayer. In this case, the system appears to have mostly converged after approximately 80 ns of simulation time. It still undergoes fluctuations in the order of a few Å2. These fluctuations increase with the fluidity of the bilayer, e.g. with a decrease in the saturation of acids or an increase in temperature.

The APL can also be used to compare simulation results to experimental values. This is most reliable for single-component mixtures and not a trivial process for mixtures.

Figure 3-11. The Membrane Thickness tab in the Membrane Analysis Viewer.

  1. Switch to the Membrane Thickness tab
  2. You can shift the two dashed blue sliders via drag-and-drop to find a converged membrane thickness, the value at the bottom is updated automatically
  3. Close the Membrane Analysis Viewer panel

The membrane thickness is another parameter that is specific to membrane structure and composition. A stable thickness value that fluctuates around a mean can be used to judge the convergence of a simulation, indicating sufficient equilibration. In this case, the system appears to have mostly converged after 80 ns of simulation time. The thickness can also be used to compare simulation results to experimental values. This is most reliable for single-component mixtures and not a trivial process for mixtures.

Both the APL and membrane thickness indicate that the short relaxation that is automatically included after the assembly stage in the Complex Bilayer Builder panel is not sufficient to fully equilibrate the system. It is designed to give a suitable low-energy starting point for longer MD simulations. We always recommend running a subsequent MD simulation to check for parameter convergence. The necessary equilibration length varies with the complexity of the system and is not easily estimated a priori.

4. Building a Multi-Component Lipid Bilayer Including a Membrane Protein

In this section, we will build a two-component lipid bilayer containing the two phospholipids POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) surrounded by water.

Additionally, we will insert the structure of the beta2 adrenoceptor (PDB-ID: 4LDE). We will make use of the Complex Bilayer Builder panel.

Structure files obtained from the PDB, vendors, and other sources often lack necessary information for performing modeling-related tasks. Typically, these files are missing hydrogens, have incorrect or missing bond order assignments, charge states, side chain orientation, or are missing whole loop regions. In order to make these structures suitable for MD related workflows, the Protein Preparation Workflow is used to resolve common structural issues. The structure of our protein was obtained from the OPM database and contains information on the protein’s placement inside the membrane. The structure provided in this tutorial has already been prepared. The original structure contained an antibody bound to the protein, which was deleted during the preparation. Please see the Introduction to Structure Preparation and Visualization tutorial and the Best Practices for Protein Preparation to learn more.

Figure 4-1. The structure of the prepared protein.

  1. Import the prepared protein structure via File > Import Structures: Section_04 > 4LDE_prepared_OPM.maegz
  2. Ensure the entry 4LDE - prepared is includedthe entry is represented in the Workspace, the circle in the In column is blue and selected(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries

Figure 4-2. Loading and adjusting the protein structure.

  1. Go to Tasks > Materials > Structure Builders > Complex Bilayer Builder
  2. In the Membrane Protein section, click Import from Workspace
    • The panel successfully loaded the OPM data from the file
    • The 4LDE system is loaded and the boundaries of the membrane are shown as planes in red and blue
    • The box dimensions and the water padding are updated according to the protein detected

Note that upon import, any additional atoms present in the workspace, along with the protein atoms, will be imported into the panel. This allows you to include relevant crystallographic water molecules or ligands in the system.

  1. Set both Box dimensions to 120 Å, respectively
  2. Click Adjust Protein Position…
    • The Adjust Protein Position dialog box opens
  3. Check the Show coordinate axes option, to show the orientation of the axes
  4. Set the X-axis value for Translation to 1.00 and click Translate 5 times
    • The protein should be moved to the approximate center of the box step by step
  5. Close the Adjust Protein Position dialog box

We have to ensure that the box dimensions are large enough to prevent self-interactions with periodic copies of the system. This applies to the X and Y axes in the membrane plane, as well as the water padding along the Z axis. Approx 1 nm of space or more between protein/membrane and box boundaries on each side should generally be sufficient. If your protein has long flexible regions, the margins might need to be increased accordingly.

In our case, the available OPM data was found and the protein was placed within the bilayer accordingly. We elected to slightly adjust the protein position for demonstration purposes. If no OPM data is available, you might have to thoroughly adjust the initial placement of the protein by translating and rotating it. In general, large hydrophobic regions of the protein should be located inside the bilayer core.

If your protein has a pore, you may want to use the prehydration option. This places water via a Grand Canonical Monte Carlo approach within the pore prior to equilibration and can prevent spurious structures.

Figure 4-3. The adjusted protein structure within the bilayer boundaries.

Figure 4-4. Setting up the composition of the lipid bilayer and running the job.

  1. Below the lipid table, click Add Lipid…
    • A new entry in the lipid table is created
  2. Click on the newly created POPC entry to open the dropdown menu and change the lipid type to POPE
  3. Ensure that the bilayer composition is a 50:50 ratio of POPC to POPE
  4. Make sure the Water Padding is set to at least 33 Å
    • This defines the height of water to add above and below the membrane and depends on the dimensions of your protein
  5. Check the option to Neutralize system, as the protein is charged
  6. Change the Job Name to complex_bilayer_4LDE
  7. Adjust the job settings as needed
    • This job requires a GPU host for the MD relaxation steps after the system is assembled. It can be completed in about 1.5 hours.
  8. If you would like to run the job yourself, click Run, otherwise, import the provided results from the zip file via File > Import Structures: Section_04 > complex_bilayer_4LDE > complex_bilayer_4LDE-out.cms
  9. Close the Complex Bilayer Builder panel

Rather than creating a symmetrical composition of both leaflets, you can also construct the upper and lower leaflets of the bilayer with an asymmetric composition, featuring differences in the types and ratios of lipids. This is advanced model building and requires extra consideration of the biophysical aspects of asymmetric bilayers.

Available phospholipids are POPC, POPE, DPPC and DMPC. In addition, cholesterol (CHL) is available. Besides the provided components, you can import your own custom lipids either via SMILES or a molecular structure from the workspace.

Besides the SPC (default) and SPCE water models, several TIP water models are available. For information regarding the FF parametrization, see the OPLS Force Field documentation.

Our system consists of zwitterionic (net-neutral) lipids, water, and the protein. The latter contains charged amino acids so ions are needed to neutralize the system. The types of ions for neutralizing the system and an additional salt concentration (e.g. to mimic physiological or experimental conditions) can be specified.

Figure 4-5. Selecting the hydrophilic atoms of the lipids.

Now we will visually inspect the assembled and relaxed protein-bilayer system.

 

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue and select(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries the Assembled system entry
  2. In the top toolbar click Define…
    • The Atom Selection panel opens
  3. In the Atom tab, scroll down to and select hydrophilic, then check the option for true, and click Add
    • The box at the bottom showing the atom selection language (ASL) should be updated
    • This selects all atoms in the bilayer labeled as hydrophilic
    • It is also possible to select the hydrophobic atoms, crystallographic waters, or waters added via prehydration
  4. Now click OK
  5. Display the selected atoms in the CPK representation
    • You can see the hydrophilic atoms of the head groups, oxygen in red and nitrogen in blue

Figure 4-6. Visually inspecting the assembled and relaxed protein-lipid system.

  1. In the Quick Select section in the top toolbar, click All to select all atoms in the system
  2. In the Style dropdown menu, click show polar hydrogens only
  3. In the Quick Select section, click Water
  4. In the Style dropdown menu, choose the  ball-and-stick representation

Now you can visually inspect the system. It may be useful to hide the water molecules in order to examine the proteins positioning inside the membrane a bit better.

In a correctly assembled and relaxed lipid bilayer, the lipid headgroups should not be present near the center of the bilayer and the tails should point towards the inside. Any packing defects in the bilayer system after the initial relaxation can usually be resolved by additional equilibration via MD simulations, but this should be an unlikely event.

Unless you elect to prehydrate a protein pore, there should not be a large number of water molecules forming a slab within the bilayer or a pore through the bilayer. If a few individual water molecules are found within the membrane, this can usually be resolved through further MD equilibration.

In this system, there are no lipid head groups near the centre of the bilayer and no lipid tails sticking out. Additionally, only a single water molecule can be found inside the bilayer next to one of the helices.

Determining the orientation and positioning of a membrane protein is not a trivial task, so we recommend checking for experimental data to guide your selection. If no data is available, the structure of the protein can provide hints for its position: In general, large hydrophobic regions of the protein should be located inside the bilayer core. Achieving a properly equilibrated positioning in this scenario however calls for much longer simulation times than presented in this tutorial.

5. Conclusion and References

In this tutorial, we learned how to create a multi-component lipid bilayer and how to embed a membrane protein using the Complex Bilayer Builder panel. Additionally, we calculated typical biophysical properties of a bilayer, such as area per lipid and bilayer thickness, with the Membrane Analysis panels.

For further learning:

For introductory content, focused on navigating the Schrödinger Materials Science interface, an Introduction to Materials Science Maestro tutorial is available. Please visit the materials science training website for access to 100+ tutorials. For scientific inquiries or technical troubleshooting, submit a ticket to our Technical Support Scientists at help@schrodinger.com.

For self-paced, asynchronous, online courses in Materials Science modeling, including access to Schrödinger software, please visit the Schrödinger Online Learning portal on our website.

For some related practice, proceed to explore other relevant tutorials:

6. Glossary of Terms

Entry List - a simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

Included - the entry is represented in the Workspace, the circle in the In column is blue

Project Table - displays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data

Recent actions - This is a list of your recent actions, which you can use to reopen a panel, displayed below the Browse row. (Right-click to delete.)

Scratch Project - a temporary project in which work is not saved, closing a scratch project removes all current work and begins a new scratch project

Selected - (1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries

Working Directory - the location where files are saved

Workspace - the 3D display area in the center of the main window, where molecular structures are displayed