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Supplemental Experimental Procedures

Molecular Dynamics Simulations

MD simulations were performed using a modified version of GROMACS 4.6 (Hess et al.,

2008). We used the AMBER99sb force field (Hornak et al., 2006) and SPC/E water model for

the equilibrium and production simulations. Parameters for ions and lipids are derived from

(Joung and Cheatham, 2008; Berger et al., 1997). Short-range electrostatic interactions were

calculated with a cutoff of 1.0 nm, whereas long-range electrostatic interactions were treated

by the particle mesh Ewald method (Darden et al., 1993; Essmann et al., 1995). The cutoff

for van der Waals interactions was set to 1 nm. The simulations were performed at 300 K

with a velocity rescaling thermostat (Bussi et al., 2007). The pressure was kept at 1 bar by

means of a semi-isotropic Berendsen barostat (Berendsen et al., 1984). All bonds were

constrained with the LINCS algorithm (Hess et al., 1997). Using virtual sites for hydrogen

atoms allowed simulations to be performed with a 4 fs integration time step (Feenstra et al.,

1999). Crystallographic structures of TRAAK including closed and open membrane opening

conformations (PDB ID code: 4I9W (Brohawn et al., 2013)) were adopted to generate the

initial configuration in the MD simulations. Missing atoms and loops in the crystal structure

were modeled using the program loopy (Sato et al., 2008). The dimeric protein without the

antibody antigen-binding fragments was embedded into an aqueous POPC lipid bilayer with

0.6 M KCl using the GROMACS tool g_membed (Wolf et al., 2010). The system was

equilibrated for 20 ns with position restraints on all heavy atoms using a force constant of

1000 kJ mol-1 nm-2 to the reference structure, followed by an additional 10 ns simulation

without position restraints.

For the computational electrophysiology study (Kutzner et al., 2011), the equilibrated system

was duplicated along the z direction and transmembrane potential gradients were generated

by introducing a charge difference of 2 K+ ions between the two compartments separated by

the two lipid bilayers. During the MD simulations, the number of the ions was kept constant

by an additional algorithm (Kutzner et al., 2011). The resulting membrane potential can be

calculated by double-integration of the charge distribution using the Poisson equation as

implemented in the GROMACS tool g_potential (Tieleman and Berendsen, 1996). In the

production simulations, two ionic configurations were employed in the starting structures:

KWKKK and KKKKK (S0, S1, S2, S3, S4), respectively. During the simulations a permeation

event was counted when an ion moved from the cavity to the filter, and another ion left the

S0 position.

References

Hess, B., Kutzner, C., van der Spoel, D., and Lindahl, E. (2008). GROMACS 4: algorithms forhighly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput 4,435–447.Hornak, V., Abel, R., Okur, A., Strockbine, B., Roitberg, A., and Simmerling, C. (2006).Comparison of multiple Amber force fields and development of improved protein backboneparameters. Proteins 65, 712-725.Joung, I.S., and Cheatham, T.E., 3rd. (2008). Determination of alkali and halide monovalention parameters for use in explicitly solvated biomolecular simulations. J Phys Chem B 112,9020–9041.Berger, O., Edholm, O., and Jähnig, F. (1997). Molecular dynamics simulations of a fluidbilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constanttemperature. Biophys J 72, 2002–2013.Darden, T., York, D., and Pedersen, L. (1993). Particle mesh Eward: An N log(N) method forEwald sums in large systems. J Chem Phys 98, 10089.Essmann, U., Perera, L., Berkowitz, M. L., Darden, T., Lee, H., and Pedersen, L. G. (1995).A smooth particle mesh Ewald method. J Chem Phys 103, 8577-8593.Bussi, G., Donadio, D., and Parrinello, M. (2007) Canonical sampling through velocityrescaling. J Chem Phys 126, 014101.Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., Dinola, A., and Haak, J. R.(1984). Molecular dynamics with coupling to an external bath. J Chem Phys 81, 3684-3690.Hess, B., Bekker, H., Berendsen, H. J. C., and Fraaije, J. G. E. M. (1997). LINCS: A linearconstraint solver for molecular simulations. J Comput Chem 18, 1463-1472.Feenstra, K. A., Hess, B., and Berendsen, H. J. C. (1999). Improving efficiency of large time-scale molecular dynamics simulations of hydrogen-rich systems. J Comput Chem 20, 786-788.Brohawn, S. G., Campbell, E. B., and MacKinnon, R. (2013). Domain-swapped chainconnectivity and gated membrane access in a Fab-mediated crystal of the human TRAAK K+channel. Proc Natl Acad Sci USA 110(6), 2129-2134.Sato, C. S., Fasnacht, M., Zhu, J., Forrest, L., and Honig, B. (2008). Loop modeling:Sampling, filtering, and scoring. Proteins 70(3), 834-843.Wolf, M.G., Hoefling, M., Aponte-Santamaría, C., Grubmüller, H., and Groenhof, G. (2010).g_membed: Efficient insertion of a membrane protein into an equilibrated lipid bilayer withminimal perturbation. J Comput Chem 31, 2169–2174.Kutzner, C., Grubmüller, H., de Groot, B.L., and Zachariae, U. (2011). Computationalelectrophysiology: the molecular dynamics of ion channel permeation and selectivity inatomistic detail. Biophys J 101, 809–817.Tieleman, D., and Berendsen, H. J. C. (1996). Molecular dynamics simulations of a fullyhydrated dipalmitoylphosphatidylcholine bilayer with different macroscopic boundaryconditions and parameters. J Chem Phys 105, 4871–4880.

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