RMSDs from the two TMD simulations.

<p>For each simulation, the heavy-atom RMSDs for the EL4 and IL2 loops and the entire protein with respect to the target crystal structure are shown. (<b>A</b>) Results for the TMD_O→I simulation. (<b>B</b>) Results for the TMD_I→O simulation.</p

Free energy profile of AdK conformations.

<p>The free energy was defined along a conformational pathway (see Methods), and calculated from the umbrella-sampling simulations. The plotted error bars are for the free energy difference with respect to the first umbrella window at <i>α</i> = 0, estimated from the statistical uncertainties in the mean coordinate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068023#pone.0068023-Zhu2" target="_blank">[40]</a>. The green and red dashed lines indicate the projected locations of the open and closed crystal structures, respectively.</p

Some typical salt bridges in the closed (A) and open (B) AdK conformations.

<p>The two snapshots were taken from simulations C5 and O1, respectively. The images were rendered using the VMD software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068023#pone.0068023-Humphrey1" target="_blank">[45]</a>.</p

Simulation system.

<p>The protein Mhp1 (in the IF conformation) is embedded in a POPE lipid bilayer.</p

A comparison of the average conformations from the umbrella-sampling simulations to the crystal structures.

<p>Average C<i>_α</i> coordinates for each of the 30 umbrella windows were calculated from the trajectories. The RMSDs between these average C<i>_α</i> coordinates and the two crystal structures are plotted in the figure.</p

Comparison of the OF [14] and IF [15] crystal structures of Mhp1.

<p><b>(A)</b> A superimposition of the two crystal structures, with the extracellular side up. The EL4 extracellular loop (between the OUT7-8 helix and TM8) and the IL2 intracellular loop (between TM4 and TM5) are highlighted. <b>(B)</b> The backbone structures for the EL4 and IL2 loops in the OF and IF conformations. The side chain of Ile161 in the IL2 loop is also shown. Two distances, between S295:H and V291:O in the EL4 loop and between I164:H and G160:O in the IL2 loop, are labeled. <b>(C)</b> Difference in the backbone torsions. For each residue, the differences in the backbone φ and ѱ angles between the OF and IF crystal structures are calculated, and the combined deviation </p><p></p><p></p><p></p><p></p><p></p><p></p><p><mo stretchy="false">(</mo><mi>Δ</mi><mi>φ</mi><mo stretchy="false">)</mo></p><mn>2</mn><p></p><mo>+</mo><p></p><p><mo stretchy="false">(</mo><mi>Δ</mi><mi>ψ</mi><mo stretchy="false">)</mo></p><mn>2</mn><p></p><p></p><p></p><p></p><p></p><p></p> is plotted.<p></p

Thickness of the lipid bilayer.

<p>The thickness was calculated as the difference between the average <i>z</i> coordinates (membrane plane normal to the <i>z</i> axis) of the lipid phosphorus atoms in each leaflet. (<b>A</b>) Results for the unbiased simulation with Mhp1 in the OF state. (<b>B</b>) Results for the IF-state simulation.</p

The simulation system.

<p>AdK was initially in the closed conformation in this particular simulation. The AMPbd, LID, and CORE domains of the protein are colored red, yellow, and blue, respectively. K+ and Cl⁻ ions are drawn as blue and red spheres, respectively. The image was rendered using the VMD software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068023#pone.0068023-Humphrey1" target="_blank">[45]</a>.</p

RMSDs between the average protein conformations from the unrestrained simulations and the AdK crystal structures.

<p>The protein conformation (represented by its C<i>_α</i> coordinates) from each frame in the simulation trajectories was aligned against the crystal structure, and the mean conformation for each simulation was obtained by averaging the aligned C coordinates over the frames in the last 40 ns of the simulation. These mean conformations were compared to the open (4AKE) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068023#pone.0068023-Muller2" target="_blank">[7]</a> and the closed (1AKE) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068023#pone.0068023-Muller1" target="_blank">[6]</a> AdK crystal structures, with the RMSDs provided in the table.</p

Conformational transition in the isolated EL4 loop.

<p><b>(A)</b> The φ and ѱ angles for residues Gly292, Gly293 and Val294 along the transition pathway, with the color representing the progression of the transition, from <i>blue</i> for the OF state to <i>red</i> for the IF state. <b>(B)</b> The calculated free energy profile for the transition. The <i>blue</i> and <i>red</i> curves (which are largely overlapping) were obtained from two independent groups of string-method simulations with different initial coordinates. <b>(C)</b> Some snapshots along the transition pathway between the OF and the IF states, including the backbone structures at the major energetic barriers identified in (B). Major torsion angles involved in each barrier are indicated.</p