Cryo-EM: Spinning the Micelles Away

Structural characterization of integral membrane proteins (MPs) demands that the samples be pure, monodisperse, and stable. Detergents are required to extract MPs from the lipid bilayer in which they reside and to stabilize them for downstream biophysical analyses. Some of the best MP-stabilizing detergents pose problems for cryo-EM studies, but in this issue of Structure, Hauer et al. (2015) now offer a solution called GraDeR

Can Shaker Potassium Channels be Locked in the Deactivated State?

For structural studies it would be useful to constrain the voltage sensor of a voltage-gated channel in its deactivated state. Here we consider one Shaker potassium channel mutant and speculate about others that might allow the channel to remain deactivated at zero membrane potential. Ionic and gating currents of F370C Shaker, expressed in Xenopus oocytes, were recorded in patches with internal application of the methanethiosulfonate reagent MTSET. It appears that the voltage dependence of voltage sensor movement is strongly shifted by reaction with internal MTSET, such that the voltage sensors appear to remain deactivated even at positive potentials. A disadvantage of this construct is that the rate of modification of voltage sensors by MTSET is quite low, ∼0.17 mM−1·s−1 at −80 mV, and is expected to be much lower at depolarized potentials

Methods for Cryo-EM Single Particle Reconstruction of Macromolecules having Continuous Heterogeneity

Macromolecules change their shape (conformation) in the process of carrying out their functions. The imaging by cryo-electron microscopy of rapidly-frozen, individual copies of macromolecules (single particles) is a powerful and general approach to understanding the motions and energy landscapes of macromolecules. Widely-used computational methods already allow the recovery of a few distinct conformations from heterogeneous single-particle samples, but the treatment of complex forms of heterogeneity such as the continuum of possible transitory states and flexible regions remains largely an open problem. In recent years there has been a surge of new approaches for treating the more general problem of continuous heterogeneity. This paper surveys the current state of the art in this area.Comment: 20 pages, 2 figure