This tutorial will introduce you to most of the essential functionalities of the Simmune Modeler, CellDesigner and Simulator.
In part one you will learn how to create a model specifying molecular interactions and multi-molecular complexes along with associations, dissociations and transformation those complexes can undergo.
In part two using the CellDesigner for the generation of spatial compartments and cell morphologies will be illustrated.
Part three shows how to load the signaling model created in step one and the geometry defined in step two into a simulation and explore various aspects of the behavior of the model.
G protein coupled receptor (GPCR) signaling is one of the most important mechanisms in cell physiology, playing essential roles for phenomena as diverse as vision, pathogen sensing in the immune system and directed cell migration (chemotaxis). Ligation of the extracellular domain of the receptor makes the cytosolic part of the receptor available for interaction with heterotrimeric G proteins, consisting of three subunits, Galpha, Gbeta and Ggamma. Gbeta and -gamma are thought to form a stable complex, Gbetagamma but the strength of association with the Galpha subunit depends on whether Galpha is bound to guanosine triphosphate (GTP) or -diphosphate (GDP). The activated receptor can catalyze the GDP -> GTP exchange at Galpha, causing a rapid dissociation of Gbetagamma from Galpha. Free Gbetagamma and GTP-Galpha interact with signaling molecules in the cytoplasm or at the inner membrane leaflet, thereby propagating the receptor ligation signal into the cell. Galpha has an "auto-GTPase" activity, allowing it to deactivate itself and then reassociate with Gbetagamma and start the cycle of receptor catalyzed activation again.
Start the Simmune Modeler. Select "Model"->"New".
Specify a name for the model (e.g. G_proteins.dbf).
View of the empty model.
Click on "Molecule" to open the molecule editing page.
Click "New" and then "Add Component".
Resize the molecule component to make it look like this. Click OK.
With the first component ("Molecule component 1") selected, click "Add Component" again. Resize it according to the screenshot. Click OK.
You can move the second component to make the molecule look like this.
Add one binding site to each of the two components. (Select component, click "Add Binding Site".)
The molecule should now look like this.
Now select the Molecule -- either by clicking on the "Molecule:..." line in the "Molecule Composition" tree view or by clicking in the display outside of any of the two components. Set the molecule properties: Type: Protein; select "Membrane Associated"; Category: Receptor; set Diff_Coeff to 0.001 square microns per second (the diffusion coefficient of a large transmembrane receptor) and specify a name ("Receptor").
Select the Molecule component 1, select as Location "Inside-membrane", set the name to "Intracellular Domain" (or something similar).
Select the Molecule component 2, select as Location "Outside-membrane", set the name to "Extracellular Domain" (or something similar). These two steps specify that the receptor is inserted into the cellular membrane with component 2 facing the extracellular space and component 1 facing the cytoplasm.
Now, we will add a state to the Intracellular domain that encodes the information of the activation state of the receptor. Select the component 1, then click "Add/Modify/Remove" next to the "States" text line. A small window should open.
Enter a name for the state (e.g. "activated") then click "Add". Answer the question about changing the color of the tag representing the new state with "Yes". Note: In models in which a molecular component can assume multiple different states, for instance Tyrosine / Threonine phosphorylations etc., the use of multiple colors for the different states facilitates identifying the various possible combinations in the Modeler interface.
Select a color. The click OK.
The "Molecule Component States" window should now look like this. Click "Done".
Click "Save" and "Clear". The molecule editing page should now look like this. Note: you can adjust the size of the molecule icons in "Tools"->"Preferences".
Create a second molecule ("Ligand") with properties as shown in the screenshot (eg. size 50X50).
Create a third molecule with two binding sites and with a state "GTP" for its only component (eg. size 50X25). The state will encode whether the molecule (the G protein subunit Galpha) is in the GTP or GDP state.
Set the properties for the molecule as shown in the sceenshot. After clicking on the binding sites you can specify names related to their functionality. We will use site 1 to interact with the receptor and site 2 to interact with the Gbetagamma subunit. Important: Specify that the (only) component has as Location "Inside-membrane"! Otherwise the molecule will face the extracellular space after insertion into the cytoplasmic membrane. This property of the molecule can only be set after "Membrane Associated" has been selected as property for the whole molecule. Click "Save" and "Clear".
Create the Gbetagamma subunit with properties as shown in the sceenshot (eg. size 50X50). Important: Specify that the (only) component has as Location "Inside-membrane"! Otherwise the molecule will face the extracellular space after insertion into the cytoplasmic membrane. "Save" and "Clear".
Select the Receptor for editing (by double-clicking its name/icon in the list of molecules.) Then click "New" under "Molecule interactions" on the right.
The Receptor will appear as the first binding partner of the Molecule Interaction we will define.
Double-click the Receptor in the display, then click "Flip".
Double-click the Ligand molecule in the molecule list to set it as the second binding partner and then draw a line from the binding site at the Receptor's extracellular domain to the binding site of the Ligand.
Set a name for the interaction (optional but useful for identifying reactions), e.g. "receptor ligation". Click "Save" and then "Back". Note: Interactions between molecular binding sites can be defined before they are used to create multi-molecular complexes or they can be defined during the process of connecting molecular complexes through associations. Assigning interactions using the full molecule interface as shown here can add a layer of control to the definition of your model. During the process of connecting molecular complexes through associations between molecular binding sites the Modeler will create new binding possibilities for pairs of binding sites that did not yet have them, after asking the user to confirm the new binding possibilities.
The molecule editing page should now look like this.
Click on "Complex" to open the complex editing page
Load the Receptor complex species and click "New" under "Relations". Select "Create/Edit Association (Current Complex As Component)", click "Go". Flip the receptor.
Double-click the "Species: Ligand" entry in the complex species list on the right. Then draw a line from the binding site at the Receptor's extracellular domain to the binding site of the Ligand. Click "Define Result Complex Species".
Move the Receptor and Ligand together until they touch at the binding sites. The Modeler will make a link between the sites. Specify a name for the complex species (e.g. "Ligated Receptor"). Click "Save and Back".
In the Result Complex, double-click the activated State of the Intracellular Domain and set it to "on". This means that the ligation of the receptor by the ligand will produce a receptor for which the activated state is on (Active).
Assuming that the ligand binds an inactive receptor, set the the "activated" State of the Intracellular Domain to "off".
Specify a name for the inactive Receptor (e.g. "Rec inactive") then click "Save Complex". Important note: Simmune distinguishes between "Complex Species" and "Complexes". The Complex Species outlines the molecular structure and the possible states of the Complexes belonging to the Species. In other words: A member of a Complex Species with a particular set of states (among which there can be "Don't care" entries) is a Complex. All reactions are specified using Complexes: whenever you define an association, dissociation or transformation you choose the Complex Species involved in the reaction and then define the particular state the molecule components have to be in to allow the reaction to take place. The Complex Species of the reactants determine the Complex Species of the result of the reaction. To define the Result Complex you then have to specify the states of the molecule components of the Result Complex Species.
Similarly for the Ligand complex and the "Ligated Receptor". Specify a name (optional) and a rate (obligatory) for the association as shown in the screenshot. Click "Save" and then "Back".
The Complex Species page now should look like this.
Now define an association between Galpha and Gbetagamma in a similar way: load the Galpha complex species, click "New" under "Relations", then select "Create/Edit Association", click "Go" and in the Complex Association window double-click the Gbetagamma species in the complex list.
Draw a line from the Galpha's binding site 2 to the binding site of Gbetagamma. Since we did not define this interaction on the molecule level the Modeler asks us to confirm the new molecular interaction. Click "Yes". Click "Define Result Complex Species". Note: defining interactions between binding sites at the molecular level as opposed to defining them when creating complex interactions can provide an additional level of control in situations in which molecules have several binding sites and you want to make sure to use the right ones for a specific interaction.
Move Galpha and Gbetagamma together linking them at the binding sites involved in the association. Specify "Gabg" (or similar) as name for the Result Complex Species. Click "Save and Back".
Set the State for the Galpha complex component as shown in the screenshot: "GTP" to "Inactive" for Galpha as complex 1 and for Galpha as part of the Result Complex. This means that Gbetagamma will bind to Galpha only if it is in the GDP state. Use the default names for the complexes. Set a name and a rate for the association. Click "Save" and then "Back".
The Complex Species page now should look like this.
Double-click the "Ligated Receptor" species and click "New" under "Relations". Select "Create/Edit Association", click "Go".
Double-click the "Species: Gabg" entry in the complex species list on the right. Then draw a line from the binding site at the Receptor's intracellular domain to the binding site 1 of Galpha. Since we did not define this interaction on the molecule level the Modeler asks us to confirm the new molecular interaction. Click "Yes". Click "Define Result Complex Species". Move the two complexes together until they form a link at the binding sites involved in the association. Note that you can move binding sites and components relative to each other when the "Edit Option" "Change Layout" is active. To move complexes activate the option "Move". Specify a name for the new four-molecule complex, for instance "LigRec_Gabg". Click "Save and Back".
Set the "activated" state of the receptor to "on".
Set the "GTP" state of the "G_alpha" molecule to "off"
For the Result Complex, adopt the states from the reacting complexes: "Active" for the "on" state of the Receptor and "off" for the "GTP" state of Galpha (meaning that only Galpha in the GDP state and bound to Gbetagamma will associate with the Receptor). Specify a name for the Result Complex, for instance "LicRec_Gabg_GDP" and click "Save Complex". Set a name and rate for the association. Click "Save" and then "Back". Note: When the simulator builds complexes from reactions (associations, dissociations, transformations) those states that are set to "Don't care" in the Result Complex(es) are adopted automatically from the reactants. We could therefore have left the states in the Result Complex at "don't care" and would have specified the same association. However, specifying the LicRec_Gabg_GDP complex explicitly will allow us to follow the kinetics of this particular complex in the simulation.
The complex species page for the 'Ligated Receptor' species should now look like this.
Click "New" under "Relations", then select "Create/Edit Dissociation", click "Go".
Select the "Ligated Receptor" as complex to decay. Click OK.
Below "Select Binding Site Interaction to Split" select (the only) "Binding Site Interaction 1". Click "Split".
Select "Rec inactive" as Result Complex 2.
Set a name and rate for the dissociation. Click "Save" and then "Back".
Click "Clear" in the Complex Species page.
Load the "Gabg" species and click "New" under "Relations". Select "Create/Edit Dissociation", click "Go".
Set the "GTP" state of G_alpha to "off"
Split the only binding interaction. Set a name (Gabg_GDP dissoc) and rate (0.01) for the dissociation and click "Save". Note: We assume that Gbetagamma will bind tightly to Galpha in the GDP state.
Now, we will define a decay for the case when Galpha is in the GTP state. Change the Galpha GTP state in the decaying complex to "on". Set "GTP" to "Active" in the Result Complex 2 as well. Change the name (to Gabg_GTP dissoc) and rate (to 10) and click "Save". The Modeler will prompt you to choose whether to update the previously edited dissociation or create a new dissociation. Select "Create a new dissociation". Click OK. Note: We assume that Galpha in the GTP state will be released rapidly from Gbetagamma to allow both to interact with other signaling components.
Click "Save" and then "Back".
The Complex Species page for Gabg now should look like this. Make sure both dissociations appear in the "Relations" list to the right. Click "Clear".
For Galpha to be able to dissociate from the Receptor even when it is not bound to Gbetagamma, we have to define dissociations for a Receptor-Galpha complex species. Click "New" below "Complexes" and load (by double-clicking the entries in the molecule list) the Receptor and Galpha molecules.
Double-click the Receptor and flip it.
Move the Receptor and Galpha together until they bind at their binding sites "1". Click in the graph outside the molecules or activate the "Composition" tab and select "Complex Species:" line to activate editing the properties of the new species. Set "Rec_Galpha" as Short Name and click "Save".
Click "New" under "Relations", then select "Create/Edit Dissociation", click "Go".
Set the activated state of the Receptor to "off". Leaving the GTP state of Galpha at "Don't care" we configure this dissociation as a dissociation affecting all G proteins bound to an inactive Receptor.
Below "Select Binding Site Interaction to Split" select the (only) binding site interaction. Click "Split". Set a name (Rec inact Galpha dissoc) and rate (10) for the dissociation and click "Save". Note: "Don't care" states can be used to create 'patterns' that will match multiple complexes of a species (differing only with regard to the states that carry the "Don't care" tag in the pattern). Such patterns can be used to obtain, for instance, the total (summed) concentration the complexes matching the pattern. A complex with only "Don't care" states represents a 'pattern' that will match any complex of the species.
Now, set the receptor state activated to "on" and the Galpha GTP state to "off". Set the active state for the Receptor in the Result Complex. Using an implicit rule of the modeler application, we leave the GTP state for Galpha as Result Complex at "Don't Care". This means that during the implementation of this decay in the simulation this Result Complex will inherit its state from the molecule components state of the Complex to Decay -- which is 'off' for Galpha. We could in this case also have set the state of the Receptor result complex to "Don't care" to let the simulation use the state the receptor was in before the decay. Set the name to "Rec act Galpha_GDP dissoc", the rate to 0.01 and click "Save".The Modeler will prompt you to choose whether to update the previously edited dissociation or create a new dissociation. Select "Create a new dissociation". Click OK. Then "Save". Note: We assume that Galpha in the GDP binds tightly to the activated receptor.
To specify the dissociation of active Galpha from the activated Receptor, set the Galpha GTP state to "Active". Keep the same states for Receptor and Galpha in the Result complexes. Set the name to "Rec act Galpha_GTP dissoc", the rate to 10 and click "Save".The Modeler will prompt you to choose whether to update the previously edited dissociation or create a new dissociation. Select "Create a new dissociation". Click OK. Then "Back". Note: We assume that Galpha in the GTP state will be release rapidly from the Receptor to allow for interactions with 'downstream' effectors. Note that we have again used the implicit rule according to which "Don't care" states in Result complexes will in the simulation be replaced by the states of the Start Complex.
The Complex Species page now should look like this. Make sure all three dissociations appear in the "Relations" list.
Click "Clear" in the Complex Species page and load the "LigRec_Gabg" Complex Species.
Click "New" under "Relations", then select "Create/Edit Transformation", click "Go".
Select the "LigRec_Gab_GDP" as source complex.
In the result complex, set the Receptor "on" state and the "GTP" state of Galpha to "on". Save the "Result Complex" as "LicRec_Gabg_GTP". Set a name ("Receptor mediated Galpha GDP GTP exchange") and a rate (3) for the transformation. Click "Save" and then "Back".
Click "Clear" in the Complex Species page and load the "Galpha" Complex Species. Click "New" under "Relations", then select "Create/Edit Transformation", click "Go".
Set the GTP state to "on" in the source complex.
Set the GTP state to "off" in the result complex.
Set a name ("Galpha auto-GTPase") and a rate (0.3) for the transformation. Click "Save" and then "Back".
Clicking on "Model" on the second menu bar activates the Model view that allows us to inspect the entire model at a glance.
Next we will define the complexes that we'll use in our Simulation. Note that most of the complexes defined so far can not be instantiated in a simulation as they contain wildcards and are therefore not uniquely specified. Click on the "Standard View" button and scroll down to the "Complex Species" entry in the model overview. Open the "Gabg" entry by clicking on the "+" on it's left. You'll see that there are already three complexes.
Many complexes are only relevant as patterns in reaction rules. We can hide these in the model overview. Click on the "Display Hidden Complexes" checkbox. A checkbox will appear in front of all complexes. Disable the checkbox to hide the complex. The change will take effect if you uncheck the "Display Hidden Complexes" checkbox.
Double click on any complex under the "Gabg species. Set the status of the unbound binding site to "Don't Care" and set the GTP state to "off". Note how the name of the complex changes if you change the states; if a complex is already in the database the Modeler will fetch display the the corresponding entry. Rename the complex to "Gabg_GDP_pattern". Click "Save" to save the new name. While this complex can not be used to specify some initial molecular contents of a cell (due to its "don't cares"), it will be useful when we inspect the simulation results and want to have information about the total amount of the heterotrimeric G protein in its GDP state.
Change the "GTP" state to "on" and rename the complex to "Gabg_GTP_pattern". Click "Save" to save the new name.
Similarly, change the binding state of the unbound binding site to "Unbound" and create a complex "Gabg_GTP". Do the same for the GDP variant. These are complexes that can be used to specify the initial biochemical composition of a simulated cell (since they don't contain wildcards). Click on "Model" to return to the model overview.
We'll continue creating and/or renaming complexes for initial conditions and observation during simulations. Select any complex under the G_alpha species. Create a complex that has both binding sites set to "Don't Care". Build two variants one with the "GTP" state set to off and and one with the "GTP" state set to "on". Name them "G_alpha_GDP_pattern" and "G_alpha_GTP_pattern" respectively. These patterns will match any complex containing G_alpha in its GDP/GTP state irrespective of whether they are bound to any other molecule. In particular they will also match complexes belonging to the patterns (Gabg_GDP_pattern/Gabg_GTP_pattern) defined in the previous step. The patterns in the previous step require G_betagamma to be bound to G_alpha. We don't plan to use G_alpha in our initial conditions thus we will not create complexes of G_alpha that do not contain wildcards. Click "Back" to return to the model overview once you've created both patterns.
Hide all "G_alpha-complexes" that have default names.
Create a complex that contains the inactive unbound receptor, and name it "Rec inactive unbound"
