Membrane proteins, including ion channels, became the focus of structural proteomics midway through the 20th century. Methods for studying ion channels are diverse and include structural (X-ray crystallography, cryoelectron microscopy, currently X-ray free electron lasers) and functional (e.g., patch clamp) approaches. This review highlights the evolution of approaches to study of the structure of cardiac ion channels, provides an overview of new techniques of structural biology concerning ion channels, including the use of lipo- and nanodiscs, and discusses the contribution of electrophysiological studies and molecular dynamics to obtain a complete picture of the structure and functioning of cardiac ion channels. Electrophysiological studies have become a powerful tool for deciphering the mechanisms of ion conductivity and selectivity, gating and regulation, as well as testing molecules of pharmacological interest. Obtaining the atomic structure of ion channels became possible by the active development of X-ray crystallography and cryoelectron micro-scopy, and, recently, with the use of XFEL.

Mul'tirezistentnye shtammy Klebsiella pneumoniae yavlyayutsya odnoj iz samyh ser'eznyh prichin vnutribol'nichnyh infekcij, vyzvannyh bakteriyami, ustojchivymi k antibiotikam. Sushchestvuyut razlichnye varianty bor'by s etoj ugrozoj, odin iz nih — klinicheskoe ispol'zovanie bakteriofagov. Cel'yu raboty bylo vydelit' i detal'no oharakterizovat' virulentnyj baktriofag, imeyushchij potencial dlya terapevticheskogo primeneniya. Ispol'zovali standartnye metody fagovoj biologii, bioinformatiki, vklyuchaya sovremennye sposoby predskazaniya belkovyh struktur (programma AlphaFold), elektronnuyu mikroskopiyu. Iz obrazcov stochnyh vod byl vydelen virulentnyj podovirus KPPK108.1, otnosyashchijsya k rodu Drulisvirus, specifichno inficiruyushchij shtammy K. pneumoniae, imeyushchie kapsulyarnyj polisaharid tipa KL108, opredelena posledovatel'nost' ego genoma, issledovany ego biologicheskie svojstva i dana geneticheskaya harakteristika.

Multidrug-resistant Klebsiella pneumoniae strains are one of the major causes of nosocomial infections caused by the antibiotic-resistant bacteria. There are different options for dealing with this threat, among which is the clinical application of bacteriophages. The study was aimed to isolate and describe a virulent bactriophage, having the potential for therapeutic use. The standard phage biology and bioinformatic methods were used, which included the advanced techniques for protein structure prediction (AlphaFold software), and electron microscopy. The virulent podovirus KPPK108.1, being the member of genus Drulisvirus, which is able to specifically infect the K. pneumoniae strains with the KL108 type capsular polysaccharide, has been isolated from the wastewater. The sequence of the bactriophage genome has been defined, the biological properties have been investigated, and the genetic features have been described.

Styrene-maleic acid (SMA) copolymers are used to extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding SMA lipid particles (SMALPs). SMALPs can serve as stable water-soluble nanocontainers for structural and functional studies of membrane proteins. Here, we used SMA copolymers to study full-length pore-forming α-subunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an α-subunit hKCNQ1 and its regulatory transmembrane KCNE1 β-subunit (hKCNE1-hKCNQ1) with added affinity tags, expressed in mammalian COS-1 cells. All these recombinant proteins were shown to be functionally active. Treatment with the SMA copolymer, followed by purification on the affinity column, enabled extraction of all three channels. A DLS experiment demonstrated that negative stain electron microscopy and single particle image analysis revealed a four-fold symmetry within channel-containing SMALPs, which indicates that purified hKCNH5 and hKCNQ1 channels, as well as the hKCNE1-hKCNQ1 fusion construct, retained their structural integrity as tetramers.

Voltage-gated potassium channel Kv7.1 plays an important role in the excitability of cardiac muscle. The α-subunit of Kv7.1 (KCNQ1) is the main structural element of this channel. Tetramerization of KCNQ1 in the membrane results in formation of an ion channel, which comprises a pore and four voltage-sensing domains. Mutations in the human KCNQ1 gene are one of the major causes of inherited arrhythmias, long QT syndrome in particular. The construct encoding full-length human KCNQ1 protein was synthesized in this work, and an expression system in the Pichia pastoris yeast cells was developed. The membrane fraction of the yeast cells containing the recombinant protein (rKCNQ1) was solubilized with CHAPS detergent. To better mimic the lipid environment of the channel, lipid–protein nanodiscs were formed using solu- bilized membrane fraction and MSP2N2 protein. The rKCNQ1/nanodisc and rKCNQ1/CHAPS samples were purified using the Rho1D4 tag introduced at the C-terminus of the protein. Protein samples were examined using transmission electron microscopy with negative staining. In both cases, homogeneous rKCNQ1 samples were observed based on image analysis. Statistical analysis of the images of individual protein particles solubilized in the detergent revealed the presence of a tetrameric structure confirming intact subunit assembly. A three-dimensional channel structure reconstructed at 2.5-nm resolution represents a compact density with diameter of the membrane part of ~9 nm and height ~11 nm. Analysis of the images of rKCNQ1 in nanodiscs revealed additional electron density corresponding to the lipid bilayer fragment and the MSP2N2 protein. These results indicate that the nanodiscs facilitate protein isolation, purification, and stabilization in solution and can be used for further structural studies of human Kv7.1.

A procedure was developed for the preparation of a stalled elongation complex of Escherichia coli RNA polymerase with the nucleosome for investigation by cryo-electron microscopy. We purified the complex from the excess of free RNA polymerase and unproductive complexes on heparin resin and concentrated it on an affine monolayer formed by lipids bound to Ni ions. The use of affinity grids with an immobilized lipid monolayer helps to prevent aggregation of the particles on the grid surface. This technique can be used in the future to obtain a three-dimensional reconstruction of the EC+39 elongation complex.

Knowledge of the chromatin transcription intermediate structures can shed light on the functioning of RNA polymerase in the cell, which is important both for basic science and for the development of therapeutic approaches for the treatment of diseases associated with transcription disorders. In this study, we used affinity monolayers formed by lipids bound to Ni to concentrate RNA polymerase from a dilute solution containing glycerol. It has been shown that RNA polymerase can be isolated and concentrated on a Ni-lipid monolayer from solutions containing cryoprotectants, which usually make it difficult to visualize samples by electron microscopy. This method of isolation allowed us to obtain more diverse spatial orientations of molecules on carbon film, which facilitates three-dimensional reconstruction from TEM data. The results obtained can be used in further structural studies of the transcription processes to identify interactions in transcriptional complexes containing RNA polymerase.

A promising and reliable method for the analysis of macromolecule structures is capturing images using cryo-electron transmission microscopy and consequently performing three-dimensional reconstructions. In this study, using cryo-electron microscopy, we analyzed the structure of the complex, formed by RNA polymerase stalled at position +42 during its transcription through the nucleosome. We obtained both projection images and a three-dimensional structure of the EC-42 complex at 2.5 nm resolution. This allowed us to confirm the conformational integrity of the nucleosome during the passage of the RNA polymerase.

A fluorescent complex comprising semiconductor nanocrystals (CdTe quantum dots) and a potassium channel blocker, agitoxin (AgTx), is suggested for investigating the allocation of potassium channels (such as K-v1x) in neurons and myelinated and unmyelinated nerve fibers. It is shown that the distribution of potassium channels (K-v1x) on a neuron membrane, as well as that on myelinated and unmyelinated nerve fibers, is irregular. It is proven using the competitive binding of the AgTx-CdTe complex and other potassium channel blockers that complex binding with potassium channels on a neuron membrane is reversible and specific. This method makes it possible to investigate the allocation of channels on an isolated neuron membrane, as well as that on a membrane of a neuron connected with other neurons in vivo. The inhomogeneous allocation of potassium channels due to their morphological features and functioning is suggested.

Three-dimensional structure of the human voltage-gated channel Kv10.2 has been elucidated for the first time using the method of electron microscopy with 2.5 nm resolution. The molecule has a distinct domain structure. For interpretation of the structure, homology modeling was used with the cAMP-dependent channel MlotiK1 (C-subunit) structure used as a template for a membrane part of the channel, homology with the structure of the human potassium channel herg (A subunits) was used for the cytoplasmic subdomains PAS-PAC, and for the cNBD domain homology with the MloK1 channel was used. The homologous transmembrane part corresponds by size to the upper part of the three-dimensional reconstruction. Cytoplasmic domains of the Kv10.2 channel form the structure built according to the ‘hanging gondola’ type that is connected with the transmembrane part of the channel by linkers. The length of linkers suggests the possibility of contacts between the C-terminal cNBD domains and N-terminal PAS-domains.

A fluorescence assay to check the folding of potassium Kv channels expressed in vitro has been developed. For this aim, the fluorescently labeled channel blocker, recombinant agitoxin of yellow scorpion was employed. The level of expression of various Kv channels in vitro has been tested. It has been demonstrated that Kv2 channels form clusters on the cell surface, which are not associated with actin filaments. On the other hand, Kv10 channels form larger clusters, which are associated with actin, indicating the principal differences in the organization of cytoplasmic domains of Kv2 and Kv10 channels.