tion used for the hydrophilic loops correspond, at most, to an energetically reasonable conformation. Indeed, loops have been included for the sole purpose of providing a rough estimate of the elastic coupling, viscosity and long–range electrostatic contribution of their respective charged residues to the structure and dynamics of the TM domain. MD simulations in a membrane environment The model of the full Cx32 connexon was inserted in a pre– stabilized membrane patch of palmytoil phosphatidyl choline (POPC) phospholipids; see reference [71] for details on the parameterization and stabilization of the bilayer. Phospholipid molecules within 0.2 nm of any protein atom were removed (173 in total). The protein–membrane system was solvated with 36699 TIP3P water molecules generating a simulation box of 11.7 nm611.7 nm612 nm. Rectangular periodic boundary con- ditions were used throughout. These dimensions yielded a distance between consecutive connexon images on the membrane plane comparable to that observed in real connexon plaques measured by atomic force microscopy [72]. Twenty-nine neutralizing Cl2 counterions and fifteen K+ Cl2 pairs were added to the solution, yielding an approximately physiological salt concentration. The whole system underwent energy minimization and 1.7 ns of MD stabilization. Temperature and pressure were kept constant using Berendsen’s thermostat and barostat [73] at 300 K and 1 atm, respectively. Particle Mesh Ewald summation [74] was used for long range electrostatic interactions with a cut off of 0.8 nm for the direct interactions. Loose harmonic constraints of 0.1 kcal/ mol2 A˚2 were applied to the N and P atoms of the phospholipids heads during the first 0.2 ns. Harmonic constraints of 0.5 kcal/ mol2 A˚2 were applied to the Ca atoms of the TM domain of the proteins to avoid spurious distortions during the initial thermal- ization and pressurization of the system. Thereafter these constraints were linearly reduced to zero during the first 0.5 ns. Then, 1 ns of unconstrained MD was performed to fully stabilize the system. Finally, 12 ns of MD were carried out and used for analysis. Calculated properties The TM domain of Cx32 was defined as the collection of the following segments: residues from 19 to 40, 76 to 96, 131 to 152 and 189 to 209. Positional root mean square deviations (RMSD) were calculated on the Ca atoms from the starting configuration of the solvated system. The salt bridges reported in Table 1 were considered within a cutoff distance of 0.35 nm between acceptor and donor and 120 degrees for the donor–H–acceptor angle. Since the time–evolution of each connexin is not constrained by symmetry operations, small structural and dynamic deviations are expected from one polypeptide chain to another. However, since all proteins composing the channel are indistinguishable, quanti- ties reported in this article were calculated as averages over the MD trajectory and over the six proteins. The structure regarded as ‘‘final’’ was calculated as the time average over the last 5 ns of the MD trajectories and as the spatial average over the TM region of the six connexins. The structure resulting from the averaging was optimized by EM. A similar procedure was applied to derive an average connexin dimer, which was used to calculate the interface residues between contiguous connexins at the Protein–Protein interaction server available at http://www.biochem.ucl.ac.uk/ References All-Atoms Connexon Model bsm/PP/server/index.html. The normalized covariance matrix of the Ca atoms in the TM domain was calculated as in [75]. The distance between OH atoms of Tyr151, corresponding to the maximum constriction of the pore, were calculated between pairs of residues in opposite M3 helices in the connexon and averaged over the three possible opposite pairs. Molecular drawings were performed with VMD [76]. Comparison with Electron Density map The Ca scaffolds of our final model and that of ref. [11] were fitted against the electron density map, kindly provided by Julio Kovacs, using Situs 2.3 ([77], http://situs.biomachina.org/), a package for fitting high resolution models into low resolution maps. Using this computational tool one is able to map the tridimensional determinants of both the map and the model to a small number of points (codebook vectors). The two sets of codebook vectors are superimposed to find the best fit, after an exhaustive search in the translational and rotational degrees of freedom. The rigid docking fit was performed using 12 codebook vectors for both Ca models as well as for the electron density map. Supporting Information Text S1 Found at: doi:10.1371/journal.pone.0002614.s001 (0.03 DOC) MB Figure S1 Reconstruction and structural relaxation of the KcsA potassium channel from its Ca atoms. Global RMS deviations vs. time calculated using the crystallographic structure as reference. The four shoulders in the curve correspond to the time points in which constraints are applied (20 ps) or increased (120, 220 and 320 ps respectively). The inset shows the RMS fluctuations calculated for each residue. The four polypeptide chains are indicated by different colors. The thick lines near the abscissa indicate the helical elements in the structure. The molecular drawings show least square superposition between the crystallo- graphic (starting) structure and the reconstructed model after 20 ps, 120 ps, 220 ps and 320 ps of simulation, respectively. Found at: doi:10.1371/journal.pone.0002614.s002 (4.34 MB TIF) Figure S2 Figures S2a and S2c to S2h. Location and interactions of residues along TM2 taken from the averaged structure of the whole connexon. Figure S2b. The exposed side chain of Trp77 is in contact with the membrane phospholipids, i.e., in good position to interact with cholesterol molecules (not present in the simulation). Shown is a representative snapshot from the MD trajectory. Found at: doi:10.1371/journal.pone.0002614.s003 (8.19 MB TIF) Acknowledgments We thank Julio Kovacs for providing us the EM map of Cx43, Roberto Bruzzone for critical reading of the manuscript and Nir Ben-Tal for useful discussions. Author Contributions Analyzed the data: SP FM FZ. Wrote the paper: SP FM. Other: Performed computer simulations: FZ. Conceived, designed and performed computer simulations and models: SP. 1. Yeager M, Nicholson BJ (1996) Structure of gap junction intercellular channels. Curr Opin Struct Biol 6: 183–192. 2. Harris AL (2001) Emerging issues of connexin channels: biophysics fills the gap. Q Rev Biophys 34: 325–472. PLoS ONE | www.plosone.org 9 July 2008 | Volume 3 | Issue 7 | e2614