Homology modelling has matured into an important technique in structural biology, significantly contributing to narrowing the gap between known protein sequences and experimentally determined structures. Fully automated workflows and servers simplify and streamline the homology modelling process, also allowing users without a specific computational expertise to generate reliable protein models and have easy access to modelling results, their visualization and interpretation. Here, we present an update to the SWISS-MODEL server, which pioneered the field of automated modelling 25 years ago and been continuously further developed. Recently, its functionality has been extended to the modelling of homo- and heteromeric complexes. Starting from the amino acid sequences of the interacting proteins, both the stoichiometry and the overall structure of the complex are inferred by homology modelling. Other major improvements include the implementation of a new modelling engine, ProMod3 and the introduction a new local model quality estimation method, QMEANDisCo. SWISS-MODEL is freely available at https://swissmodel.expasy.org.

Potassium channels are opened by ligands and/or membrane potential. In voltage-gated K+ channels and the prokaryotic KcsA channel, conduction is believed to result from opening of an intracellular constriction that prevents ion entry into the pore. On the other hand, numerous ligand-gated K+ channels lack such gate, suggesting that they may be activated by a change within the selectivity filter, a narrow region at the extracellular side of the pore. Using molecular dynamics simulations and electrophysiology measurements, we show that ligand-induced conformational changes in the KcsA channel removes steric restraints at the selectivity filter, thus resulting in structural fluctuations, reduced K+ affinity, and increased ion permeation. Such activation of the selectivity filter may be a universal gating mechanism within K+ channels. The occlusion of the pore at the level of the intracellular gate appears to be secondary.

K + efflux through K + channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel's selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K + channel. Either Mg 2+ or Ca 2+ can reduce K + efflux through MthK channels. However, Ca 2+ , but not Mg 2+ , can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca 2+ ions can partially dehydrate, enabling selective accessibility of Ca 2+ to a site at the entry to the selectivity filter. Ca 2+ binding at the site interacts with K + ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.calcium | gating | permeation | dynamics | energetics P otassium (K + ) channels are activated and opened by a variety of stimuli, including ligand binding and transmembrane voltage, to enable K + efflux and thus, modulate physiological processes related to electrical excitability, such as regulation of action potential firing, smooth muscle contraction, and hormone secretion (1). In addition, many K + channels are further controlled by a gating phenomenon known as C-type inactivation, in which K + conduction is stopped, despite the continued presence of an activating stimulus (2). The mechanisms underlying C-type inactivation in voltage-gated K + channels (Kv channels) are linked to both intracellular and extracellular permeant ion concentrations, and several lines of evidence have suggested that Ctype inactivation is associated with a conformational change near the external mouth of the K + channel pore (i.e., at the canonical K + channel selectivity filter) (3-11).In Shaker Kv channels, C-type inactivation is known to be enhanced and recovery from inactivation is slowed by impermeant cations accessing the cytoplasmic side of the channel (5, 6, 10). Enhancement of inactivation by these cations suggests a working hypothesis, in which the impermeant ion prevents refilling of the selectivity filter with K + (6). Thus, K + presumably dissociates from the filter to the external solution, and this vacancy leaves the filter susceptible to a conformational change that underlies the nonconducting, inactivated state. However, the physical basis for the relation between ion movements and C-type inactivation as well as the structural underpinnings of the mechanism remain unclear.Here, we use divalent metal cations (Mg 2+ , Ca 2+ , and Sr 2+ ) as probes of inactivation mechanisms in MthK, a model K + channel of known structure (Fig. 1) (12-14). Specifically, we analyze conduction and gating of single MthK channels by electrophysiology combined with analysis of ion and protein movements by molecular simulation. Our el...