Cryo-EM structures of Kv1.2 potassium channels, conducting and non-conducting.

We present near-atomic-resolution cryo-EM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 3.2 Å, and 2.9Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2-2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin α-Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na+ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na+ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.

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.

Structural insights into the mechanism of the sodium/iodide symporter.

The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active iodide (I-) transport into the thyroid gland, the first step in the biosynthesis of the thyroid hormones-the master regulators of intermediary metabolism. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient1,2. For nearly 50 years before its molecular identification3, NIS was the molecule at the centre of the single most effective internal radiation cancer therapy: radioiodide (131I-) treatment for thyroid cancer2. Mutations in NIS cause congenital hypothyroidism, which must be treated immediately after birth to prevent stunted growth and cognitive deficiency2. Here we report three structures of rat NIS, determined by single-particle cryo-electron microscopy: one with no substrates bound; one with two Na+ and one I- bound; and one with one Na+ and the oxyanion perrhenate bound. Structural analyses, functional characterization and computational studies show the substrate-binding sites and key residues for transport activity. Our results yield insights into how NIS selects, couples and translocates anions-thereby establishing a framework for understanding NIS function-and how it transports different substrates with different stoichiometries and releases substrates from its substrate-binding cavity into the cytosol.

Structure and analysis of nanobody binding to the human ASIC1a ion channel.

ASIC1a is a proton-gated sodium channel involved in modulation of pain, fear, addiction, and ischemia-induced neuronal injury. We report isolation and characterization of alpaca-derived nanobodies (Nbs) that specifically target human ASIC1a. Cryo-electron microscopy of the human ASIC1a channel at pH 7.4 in complex with one of these, Nb.C1, yielded a structure at 2.9 Å resolution. It is revealed that Nb.C1 binds to a site overlapping with that of the Texas coral snake toxin (MitTx1) and the black mamba venom Mambalgin-1; however, the Nb.C1-binding site does not overlap with that of the inhibitory tarantula toxin psalmotoxin-1 (PcTx1). Fusion of Nb.C1 with PcTx1 in a single polypeptide markedly enhances the potency of PcTx1, whereas competition of Nb.C1 and MitTx1 for binding reduces channel activation by the toxin. Thus, Nb.C1 is a molecular tool for biochemical and structural studies of hASIC1a; a potential antidote to the pain-inducing component of coral snake bite; and a candidate to potentiate PcTx1-mediated inhibition of hASIC1a in vivo for therapeutic applications.

Computational Methods for Single-Particle Electron Cryomicroscopy.

Single-particle electron cryomicroscopy (cryo-EM) is an increasingly popular technique for elucidating the three-dimensional structure of proteins and other biologically significant complexes at near-atomic resolution. It is an imaging method that does not require crystallization and can capture molecules in their native states. In single-particle cryo-EM, the three-dimensional molecular structure needs to be determined from many noisy two-dimensional tomographic projections of individual molecules, whose orientations and positions are unknown. The high level of noise and the unknown pose parameters are two key elements that make reconstruction a challenging computational problem. Even more challenging is the inference of structural variability and flexible motions when the individual molecules being imaged are in different conformational states. This review discusses computational methods for structure determination by single-particle cryo-EM and their guiding principles from statistical inference, machine learning, and signal processing that also play a significant role in many other data science applications.

A mitochondrial megachannel resides in monomeric F1FO ATP synthase.

Purified mitochondrial ATP synthase has been shown to form Ca2+-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition pore (mPTP) but the oligomeric state required for channel formation is being debated. We reconstitute purified monomeric ATP synthase from porcine heart mitochondria into small unilamellar vesicles (SUVs) with the lipid composition of mitochondrial inner membrane and analyze its oligomeric state by electron cryomicroscopy. The cryo-EM density map reveals the presence of a single ATP synthase monomer with no density seen for a second molecule tilted at an 86o angle relative to the first. We show that this preparation of SUV-reconstituted ATP synthase monomers, when fused into giant unilamellar vesicles (GUVs), forms voltage-gated and Ca2+-activated channels with the key features of mPTP. Based on our findings we conclude that the ATP synthase monomer is sufficient, and dimer formation is not required, for mPTP activity.

Current carried by the Slc26 family member prestin does not flow through the transporter pathway.

Prestin in the lateral membrane of outer hair cells, is responsible for electromotility (EM) and a corresponding nonlinear capacitance (NLC). Prestin's voltage sensitivity is influenced by intracellular chloride. A regulator of intracellular chloride is a stretch-sensitive, non-selective conductance within the lateral membrane, GmetL. We determine that prestin itself possesses a stretch-sensitive, non-selective conductance that is largest in the presence of thiocyanate ions. This conductance is independent of the anion transporter mechanism. Prestin has been modeled, based on structural data from related anion transporters (SLC26Dg and UraA), to have a 7 + 7 inverted repeat structure with anion transport initiated by chloride binding at the intracellular cleft. Mutation of residues that bind intracellular chloride, and salicylate treatment which prevents chloride binding, have no effect on thiocyanate conductance. In contrast, other mutations reduce the conductance while preserving NLC. When superimposed on prestin's structure, the location of these mutations indicates that the ion permeation pathway lies between the core and gate ring of helices, distinct from the transporter pathway. The uncoupled current is reminiscent of an omega current in voltage-gated ion channels. We suggest that prestin itself is the main regulator of intracellular chloride concentration via a route distinct from its transporter pathway.

Statistical modeling and removal of lipid membrane projections for cryo-EM structure determination of reconstituted membrane proteins.

This paper describes steps in the single-particle cryo-EM 3D structure determination of membrane proteins in their membrane environment. Using images of the Kv1.2 potassium-channel complex reconstituted into lipid vesicles, we describe procedures for the merging of focal-pairs of exposures and the removal of the vesicle-membrane signal from the micrographs. These steps allow 3D reconstruction to be performed from the protein particle images. We construct a 2D statistical model of the vesicle structure based on higher-order singular value decomposition (HOSVD), by taking into account the structural symmetries of the vesicles in polar coordinates. Non-roundness in the vesicle structure is handled with a non-linear shape alignment to a reference, which ensures a compact model representation. The results show that the learned model is an accurate representation of the imaged vesicle structures. Precise removal of the strong membrane signals allows better alignment and classification of images of small membrane-protein particles, and allows higher-resolution 3D reconstruction.

Removal of Vesicle Structures From Transmission Electron Microscope Images.

In this paper, we address the problem of imaging membrane proteins for single-particle cryo-electron microscopy reconstruction of the isolated protein structure. More precisely, we propose a method for learning and removing the interfering vesicle signals from the micrograph, prior to reconstruction. In our approach, we estimate the subspace of the vesicle structures and project the micrographs onto the orthogonal complement of this subspace. We construct a 2D statistical model of the vesicle structure, based on higher order singular value decomposition (HOSVD), by considering the structural symmetries of the vesicles in the polar coordinate plane. We then propose to lift the HOSVD model to a novel hierarchical model by summarizing the multidimensional HOSVD coefficients by their principal components. Along with the model, a solid vesicle normalization scheme and model selection criterion are proposed to make a compact and general model. The results show that the vesicle structures are accurately separated from the background by the HOSVD model that is also able to adapt to the asymmetries of the vesicles. This is a promising result and suggests even wider applicability of the proposed approach in learning and removal of statistical structures.

Principles of cryo-EM single-particle image processing.

Single-particle reconstruction is the process by which 3D density maps are obtained from a set of low-dose cryo-EM images of individual macromolecules. This review considers the fundamental principles of this process and the steps in the overall workflow for single-particle image processing. Also considered are the limits that image signal-to-noise ratio places on resolution and the distinguishing of heterogeneous particle populations.

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.

Directly reconstructing principal components of heterogeneous particles from cryo-EM images.

Structural heterogeneity of particles can be investigated by their three-dimensional principal components. This paper addresses the question of whether, and with what algorithm, the three-dimensional principal components can be directly recovered from cryo-EM images. The first part of the paper extends the Fourier slice theorem to covariance functions showing that the three-dimensional covariance, and hence the principal components, of a heterogeneous particle can indeed be recovered from two-dimensional cryo-EM images. The second part of the paper proposes a practical algorithm for reconstructing the principal components directly from cryo-EM images without the intermediate step of calculating covariances. This algorithm is based on maximizing the posterior likelihood using the Expectation-Maximization algorithm. The last part of the paper applies this algorithm to simulated data and to two real cryo-EM data sets: a data set of the 70S ribosome with and without Elongation Factor-G (EF-G), and a data set of the influenza virus RNA dependent RNA Polymerase (RdRP). The first principal component of the 70S ribosome data set reveals the expected conformational changes of the ribosome as the EF-G binds and unbinds. The first principal component of the RdRP data set reveals a conformational change in the two dimers of the RdRP.

SubspaceEM: A fast maximum-a-posteriori algorithm for cryo-EM single particle reconstruction.

Single particle reconstruction methods based on the maximum-likelihood principle and the expectation-maximization (E-M) algorithm are popular because of their ability to produce high resolution structures. However, these algorithms are computationally very expensive, requiring a network of computational servers. To overcome this computational bottleneck, we propose a new mathematical framework for accelerating maximum-likelihood reconstructions. The speedup is by orders of magnitude and the proposed algorithm produces similar quality reconstructions compared to the standard maximum-likelihood formulation. Our approach uses subspace approximations of the cryo-electron microscopy (cryo-EM) data and projection images, greatly reducing the number of image transformations and comparisons that are computed. Experiments using simulated and actual cryo-EM data show that speedup in overall execution time compared to traditional maximum-likelihood reconstruction reaches factors of over 300.

Automatic cryo-EM particle selection for membrane proteins in spherical liposomes.

Random spherically constrained (RSC) single particle reconstruction is a method to obtain structures of membrane proteins embedded in lipid vesicles (liposomes). As in all single-particle cryo-EM methods, structure determination is greatly aided by reliable detection of protein "particles" in micrographs. After fitting and subtraction of the membrane density from a micrograph, normalized cross-correlation (NCC) and estimates of the particle signal amplitude are used to detect particles, using as references the projections of a 3D model. At each pixel position, the NCC is computed with only those references that are allowed by the geometric constraint of the particle's embedding in the spherical vesicle membrane. We describe an efficient algorithm for computing this position-dependent correlation, and demonstrate its application to selection of membrane-protein particles, GluA2 glutamate receptors, which present very different views from different projection directions.

Quantifying the local resolution of cryo-EM density maps.

We propose a definition of local resolution for three-dimensional electron cryo-microscopy (cryo-EM) density maps that uses local sinusoidal features. Our algorithm has no free parameters and is applicable to other imaging modalities, including tomography. By evaluating the local resolution of single-particle reconstructions and subtomogram averages for four example data sets, we report variable resolution across a 4- to 40-Å range.

State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels.

Large-conductance voltage- and calcium-dependent potassium channels (BK, "Big K+") are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a "gating-ring" structure has been proposed to transduce Ca(2+) binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductance. Fluorescence resonance energy transfer (FRET) between fluorescent protein inserts indicates that Ca(2+) binding produces structural changes of the gating ring that are much larger than those predicted by current X-ray crystal structures of isolated gating rings.

Expression, purification and functional reconstitution of slack sodium-activated potassium channels.

The slack (slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na(+)-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles.

Hydration-layer models for cryo-EM image simulation.

To compare cryo-EM images and 3D reconstructions with atomic structures in a quantitative way it is essential to model the electron scattering by solvent (water or ice) that surrounds protein assemblies. The most rigorous method for determining the density of solvating water atoms for this purpose has been to perform molecular-dynamics (MD) simulations of the protein-water system. In this paper we adapt the ideas of bulk-water modeling that are used in the refinement of X-ray crystal structures to the cryo-EM solvent-modeling problem. We present a continuum model for solvent density which matches MD-based results to within sampling errors. However, we also find that the simple binary-mask model of Jiang and Brünger (1994) performs nearly as well as the new model. We conclude that several methods are now available for rapid and accurate modeling of cryo-EM images and maps of solvated proteins.

A Bayesian adaptive basis algorithm for single particle reconstruction.

Traditional single particle reconstruction methods use either the Fourier or the delta function basis to represent the particle density map. This paper proposes a more flexible algorithm that adaptively chooses the basis based on the data. Because the basis adapts to the data, the reconstruction resolution and signal-to-noise ratio (SNR) is improved compared to a reconstruction with a fixed basis. Moreover, the algorithm automatically masks the particle, thereby separating it from the background. This eliminates the need for ad hoc filtering or masking in the refinement loop. The algorithm is formulated in a Bayesian maximum-a-posteriori framework and uses an efficient optimization algorithm for the maximization. Evaluations using simulated and actual cryogenic electron microscopy data show resolution and SNR improvements as well as the effective masking of particle from background.