The two-ligand ectodomain dimer remaining symmetric in the simulations.

<p>(A) The two subunits of the ectodomain dimer, as observed at the end of one of the simulations without glycosylation (left) and at the end of the simulation with full glycosylation (right), superimposed using the Cα atoms of domains I–III for reference. The EGFR ectodomains I–III are shown in blue and red, and the EGF molecules bound to them are shown in yellow and orange, respectively. (B) Domain II maintaining the same conformation in both subunits of the two-ligand EGFR dimer. Angle <i>Φ</i> (the angle formed by Cα atoms of residues 194, 239, and 296 in EGFR, or 189, 235, and 289 in dEGFR) characterizes the bending of domain II. This angle is different in each of the two subunits of the asymmetric two-ligand dEGFR dimer (the solid and dashed black lines; PDB entry 3LTF). The average angles in the simulations of two- and one-ligand EGFR dimers (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003742#pcbi-1003742-g002" target="_blank">Figs. 2</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003742#pcbi-1003742-g003" target="_blank">3</a>, and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003742#pcbi-1003742-g005" target="_blank">5</a>; labels refer to nonglycosylated EGFR simulations 1, 2, and 3, and glycosylated EGFR simulation G) are shown for each of the two subunits in blue and red (error bars correspond to the standard deviation). The angle <i>Φ</i> is illustrated in the schematic of domains I, II, and III of EGFR on the right.</p

Ligand-membrane interaction in simulations of the two-ligand EGFR dimer.

<p>(A) Snapshots from the endpoints of the simulations. The ectodomain dimers lie down on the membrane surface in a variety of ways; in each case, however, only one of the two ligands establishes strong interactions with the membrane. (B) The free energy of each ligand's interaction with its host receptor in a two-ligand EGFR dimer (upper panels) estimated using MM/GBVI, the strength of its interaction with the membrane bilayer (middle panels) estimated in the same way, and the distance between its N-terminus and the membrane (lower panels) in three independent simulations. In the middle panels, the surface area of each ligand buried by the membrane is plotted. As shown, the membrane-facing ligand (blue) enjoys greater binding free energy, and thus higher binding affinity, than the solvent-facing one (red) due to the additional energy conferred by the membrane interaction.</p

Simulations of the one-ligand dimer.

<p>(A) The conformation of the one-ligand ectodomain dimer obtained from a simulation employing the crystal structure of the two-ligand dimer (PDB entry 3NJP; ref. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003742#pcbi.1003742-Lu1" target="_blank">[23]</a>), with the ligand removed from the red subunit, as a starting state. Domains I–IV and the EGF molecule are marked. The one-ligand dimer differs from the two-ligand dimer in the conformation of the domain IV of the red subunit. (B) The one-ligand dimer lying down on the membrane. The ligand bound to this dimer faces the membrane. Snapshots from simulations of the nonglycosylated and glycosylated dimers are shown. (C) The free energy of a ligand's interaction with its host receptor in a one-ligand EGFR dimer (upper panels) estimated using MM/GBVI, the strength of its interaction with the membrane bilayer (middle panels) estimated in the same way, and the distance between the ligand's N-terminus and the membrane (lower panels) in three independent simulations in which the receptors were not glycosylated and in one additional simulation in which they were. Also shown (middle panels) is the total surface area of the ligand buried due to its interactions with the receptor and the membrane. As indicated by these data, the additional free energy conferred by the ligand's membrane interaction is a significant fraction of its interaction energy with the receptor.</p

Simulation of fully glycosylated EGFR.

<p>(A) A fully glycosylated ectodomain dimer of EGFR. The BiS1F1, Man8, and Man6 glycans attached to EGFR are colored by atom type (gray for carbon, red for oxygen, and blue for nitrogen). (B) The conformation at the end of the simulation shown from two opposite directions. (C) Distance between the N terminus of each ligand (blue, membrane-facing ligand; red, solvent-facing ligand) and the membrane surface (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003742#s4" target="_blank">Methods</a>) in the glycosylated-EGFR simulation, and total surface area of the ligand buried due to its interactions with the receptor and the membrane (left panels). Also shown are the results of the MM/GBVI calculations of the free energy of each ligand's interaction with its host receptor in the glycosylated two-ligand EGFR dimer (middle panel) and the results of similar calculations for each ligand's interaction with the membrane bilayer (the right panel). The membrane-facing ligand enjoys greater binding free energy, and thus higher binding affinity, than the solvent-facing one due to the additional energy conferred by the membrane interaction.</p

“Staggered” and “flush” conformations of the extracellular dimers.

<p>(A) The staggered and flush conformations <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003742#pcbi.1003742-Liu1" target="_blank">[20]</a> are observed in PDB entries 1IVO and 1MOX, respectively. These two conformations are shown at the top and in the middle. At the bottom, the yellow subunits of both crystal structures are superposed and the view is from above (relative to the other two images). The conformations can be distinguished by the angle <i>θ</i> formed by the Cα atoms of Ile190 and Pro204 of one subunit and Pro204 of the other. (B) Distributions of <i>θ</i> observed in simulations of the one- (black) and two-ligand (red) EGFR dimers. Data from the simulations of ectodomains in solution, reported in ref. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003742#pcbi.1003742-Arkhipov1" target="_blank">[26]</a>, and data from simulations of nonglycosylated and glycosylated EGFR constructs with the membrane, which are reported in the present study, are shown from top to bottom, respectively. Values of <i>θ</i> from the crystal structures are indicated. Two slightly different <i>θ</i> values are obtained for each crystal structure, because the structures are not exactly symmetric; the spaces between these values are shown as colored bands.</p

Computing extracellular potential.

<p>(<b>A</b>) Schematic of the compartmental model of a cell in relationship to the recording electrode. The calculation of the extracellular potential involves computing the transfer resistances <i>R_mn</i> between each n-th dendritic segment and m-th recording site on the electrode. (<b>B</b>) Extracellular spike “signatures” of individual cells recorded on the mesh electrode (black dots), using two single-cell models from the layer 4 network model as examples: PV2 (left) and Nr5a1 (right). (<b>C</b>) Modeled extracellular recordings with the linear electrode positioned along the axis of the cylinder in the layer 4 model (left). Extracellular potential responses (right) show all simulated data (color map) as well as from six select channels (black traces superimposed on the color map).</p

Computational performance.

<p>(<b>A</b>) Scaling of wall time duration (normalized by the duration on a single CPU core) with the number of CPU cores for the simulation set up (blue circles) and run (red circles) of the layer 4 model (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201630#pone.0201630.g005" target="_blank">Fig 5</a>). The ideal scaling is indicated by the dashed line. (<b>B</b>) Wall time increase when computing the extracellular potential for both set up (blue circles) and run (red circles) durations. (<b>C</b>) Scaling of the wall time with the simulated time for a long simulation. The non-ideal scaling with the increase in the number of cores corresponds to the deviations from the dashed line in (A).</p

Running simulations.

<p>(<b>A</b>) Relationships among various elements involved in running simulations with BioNet. The pre-built network (blue), is passed to the main Python script (pink) that loads custom user modules and runs BioNet/NEURON to produce the simulation output (purple). (<b>B</b>) The stages of the simulation executed by the main Python script. (<b>C</b>) Algorithm for distributing the cells over a parallel architecture. This simple example shows 10 cells distributed across 4 parallel processes (typically each parallel process corresponds to a CPU core). Cells are assigned to each process in turn (a “round-robin” assignment).</p