Start the Simmune Modeler.

Click "Model" -> "Open" and select the model file YeastMAPK.dbf. Click "Open".

The Modeler will open the Model Summary page.

You can expand the 'Molecule' entry in the treeview to see a listing of the molecules in the model.

...or you can expand the 'Complex Species' entry to see a listing of all complex species and, in subtrees, the complexes that have been defined based on those species.

You can customize some display options of the modeler with the help of the tools->preferences menu. For instance, you can change the icon size of the icons representing molecules and complex species / complexes.

Click "Molecule" on the second menu bar to switch to the Molecule page.

Double-click the molecule icon/name Gbg in the molecule list to view its properties. The page displays the properties of the Gbg G-protein subunit in the model. The annotation gives some information about the role that this molecule plays for the model and explains, for instance, why the diffusion coefficient of this molecule is set to a very small value.

Clicking on the molecule icon or on 'molecule component1' in the 'molecule composition' window section will switch to a display of the properties of the (only) component of this molecule. For example, the location of this membrane bound molecule is specified as 'Inside-membrane'. This setting will instruct the simulator application to insert this molecule into the inner leaflet of the membrane of a simulated cell.

Double-click the molecule icon/name Ste5 in the molecule list. Ste5 in the model has four binding sites with binding partners as indicated by the (optional) names of the binding sites: Gbg, Ste11, Ste7, Fus3. In contrast to Gbg it is cytosolic, freely diffusing.

Double-click the molecule icon/name Ste11 in the molecule list. Ste11, in addition to its binding sites, has a state tag that keeps track of the phosphorylation state of Ste11.

Select "Molecule component 1" from the "Molecule Composition" tree view. It is the only component of the molecule Ste11. The component should be highlighted in the molecule graph.

Clicking on "Add/Modify/Remove" next to the "States" field displaying the state tag name for the component opens a small window showing the state "phosphorylated". The state of binding sites (whether they are bound or unbound or whether their binding status plays no role for a particular aspect of a model) is stored as a symbol for each binding site. We will return to this further below.

Note: All molecules in this model have only one component. Please have a look at the Simmune Tutorial "G protein Signaling" for a model including a multi-component transmembrane receptor.

Click on "Complex" on the second menu bar to switch to the Complex page.

Double-click Species:Ste11. The page displays information about the Complex Species Ste11, including about participation of Ste11 in reactions (Transformations (none), Associations (2), Dissociations (7).

The list below "Complex" shows the various state combinations, formed by different combinations of component tags, that play a role in the model.

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. To remind the user of this distinction, state tags in Complex Species are diagonally half filled whereas the tags in Complexes can be empty ("off"), full ("on") or with a dotted line ("Don't care").

Check the box "Display Hidden Complexes" at the bottom of the right-hand list of Relations for the Complex Species. This will display additional complexes in the Complex list that were previously not shown.

Double-click the "pSte11_unbound" entry in the Complex list. In this Complex (called "pSte11_unbound"), the phosphorylated state is set to "on", indicating that this complex represents a phosphorylated version of Ste11 (Note: the model uses simple - as opposed to dual - phosphorylation of the components of the Mating Pheromone triggered MAPK cascade.). Both binding sites are in the "Unbound" state. Visually, this binding status is indicated by empty circles.

Double-click the "MP2C_pSte11 assoc" entry in the "Relations"->"Associations" list of the Complex. The Complex Association page shows the two interacting, single-molecular complexes "Complex 1" pSte11_unbound and "Complex 2" MP2C. By requiring that all binding sites are "Unbound" we assume (and define in the model) that the phosphatase MP2C can only bind to its target, phosphorylated Ste11 when Ste11 is not bound to any other molecule, for instance the scaffold Ste5. Note that the association involves a switch of the status of binding site 2 of Ste11 from "Unbound" to "Bound". Keeping track of bound binding sites also allows us to query the model efficiently for all versions of Ste11 that are, for instance, not bound at all, or only bound at site 2 etc. without having to enumerate the various complexes that contain Ste11 in the requested binding state. We will return to this aspect below. The properties of the association furthermore include specifications of the location of the binding partners relative to a membrane ("No Orientation", "trans-binding", "cis-binding"). Both binding partners are cytosolic here (which also means that the association rate has 3D units l/(mol*s)), so that the interaction has "No Orientation" selected. (Please take a look at the E-cadherin model accompanying the manuscript "Computational modeling of cellular signaling processes embedded into dynamic spatial contexts" for situations that involve orientation issues.) Return to the Complex page for Ste11 by hitting the back button (blue arrow in the second menu bar) twice.

Click on "Complex" at the top of the page to activate the complex selection list. Then scroll down in the "Complexes" list and select the Species MP2C_Ste11, the result of the association we just took a look at. Double-click the Species.

On the complex page for MP2C_Ste11 double-click the Transformation "Ste11 dephosphorylation". The Transformation page shows that the complex consisting of the phosphatase MP2C and phosphorylated Ste11 is transformed into a complex containing non-phosphorylated Ste11. This dephosphorylation is encoded in the state switch of the 'phosphorylated' tag of Ste11. Note that the rules specifying the transformation feature a "Don't care" symbol for the state of binding site 1 of Ste11 (gray dotted line around the binding site). We could have specified an "Unbound" state here but the only molecule binding to site 1 is Ste20 and the interactions between Ste20 and Ste11 requires Ste11 to be bound to the scaffold Ste5 through binding site 2 (see below) which is already occupied by MP2C here.

Click on "Complex" at the top of the page to activate the complex selection list. Then select Ste20 and look at its interaction with Ste11. Ste11 can bind to Ste20 only when Ste11 is already bound to Ste5.

Return to the complex page and double-click the Species Fus3.Ste5.Ste7. The complex page shows the complex species consisting of the MAPKK Ste7 (yellow) bound to the MAPK Fus3 (red) and to the scaffold Ste5 (white). This complex species is the source of phosphorylated Fus3 in the model. Double-click the transformation "Fus3 phosphorylation at scaffold".

Ste7, in order to be able to phosphorylate Fus3 according to the scheme shown here, has to be phosphorylated, it is bound to Fus3 through its binding site 1 and it is bound via site 2 to Ste5, the scaffold molecule bundling the components of the pheromone response of yeast. For Fus3, on the other hand, the interaction with Ste7 uses the binding site that would allow Fus3 to interact with Ste5 ("bnd2" is Active but "Ste5_bnd" is not). This activation scheme for Fus3 was used, instead of the more classical view that both Ste7 and Fus3 should be directly Ste5-bound, based on the report by Good, M. et al "The Ste5 Scaffold Directs Mating Signaling by Catalytically Unlocking the Fus3 MAP Kinase for Activation" (Cell, 2009 Mar 20;136(6):1085-97) and based on the constraints given by the measurements of Fus3 phosphorylation and complexation with Ste5 given in Maeder C.I. et al "Spatial regulation of Fus3 MAP kinase activity through a reaction-diffusion mechanism in yeast pheromone signalling" (Nat Cell Biol. 2007 Nov;9(11):1319-26). See the Supplementary Text 5 ("Discussion of experimentally provided constraints for computational modeling of the yeast pheromone response") and the discussion in the section 3 of this text.

Finally, we will take a look at some of those complexes that will be used to explore the behavior of the model when it is simulated with the simulator. The complex 'scaffold_bnd_pSte7_Fus3_unphos' that is the start complex in the transformation "Fus3 phosphorylation at scaffold" is one of them. As discussed above, the state tag specifying the state of Ste7 in this complex requires that Ste7 be active (phosphorylated) in addition to being bound to Ste5. The binding state of Ste5 itself, however is not specified. This means that this two-molecule complex pattern will match any complex containing phosphorylated Ste7, unphosphorylated Fus3 bound to Ste7 and Ste5. As will be shown below, this applies to 28 different complexes for which we can query the total (summed) concentration in the different regions of the simulated cell simply by querying the concentration of this pattern.

Return to the complex page and double-click the Species Ste5_Ste7. This complex species consists of Ste7 bound to the scaffold Ste5. Its complex Ste5_Ste7_all will match any complex containing Ste7 bound to Ste5. Similar patterns are defined for Ste11 bound to Ste5 (Ste5_Ste11_all) and Fus3 bound to Ste5 (Ste5_Fus3_all).