From June 8 to 11, 2025, the 5th Russian International Conference on Cryo-Electron Microscopy (RICCEM-2025) was held at the Faculty of Biology at Moscow State University. More than 300 researchers from 11 countries participated in online and offline formats. This article briefly discusses the contents of the articles in the special issue of the journal “Vestnik Moskovskogo universiteta. Seria 16. Biologia” published following the conference.

Microtubules are the basic elements of the cytoskeleton of eukaryotic cells. Due to their multifunctionality, unique structure and high mechanical rigidity, they remain a favorite object of research using various microscopy techniques, including cryo-electron microscopy. Despite impressive advances in visualizing the microtubule lattice, flexible elements of their structure – individual protofilaments at the assembling or disassembling ends, as well as regulatory unstructured peptides known as C-terminal “tails” – are still poorly visualized. In this paper, we discuss progress in the application of cryo-electron microscopy and tomography to the study of these structural elements, as well as the role and potential of molecular modeling methods for the analysis and interpretation of the obtained experimental data.

Progress in fundamental research is directly related to the emergence of new research methods that not only expand the established classical concepts, but can provide information that fundamentally changes them. According to our data, vimentin filaments, binding to mitochondria, determine their distribution and mobility in cells, affect the level of their membrane potential, there is a region responsible for the interaction of vimentin filaments with mitochondria in the N-terminal part of the vimentin molecule and similar amino acid sequences are found in other proteins, for example, in desmin. Since direct interaction of vimentin filaments with microtubules and actin filaments has already been shown, these facts together allowed us to assume that the connection of individual cytoskeleton components with each other and with mitochondria is not limited to interaction through cross-linker and motor proteins. Vimentin (and possibly other intermediate filament proteins) can regulate cytoskeletal interactions with mitochondria. A revolutionary study performed using cryo-electron tomography and fundamentally changing our understanding of the three-dimensional structure of vimentin filaments motivated us to use the capabilities of the cryo-electron microscopy method to try to identify the binding sites of vimentin with other cytoskeletal components and mitochondria. To meet this challenge is fundamentally possible if we combine super-resolution microscopy and cryo-electron tomography, which will bridge the existing “resolution gap” and solve associated problems.

Erythrocyte-derived extracellular vesicles (EDEVs) are a promising tool for “targeted drug delivery,” and this study aimed to compare several methods of obtaining EDEVs from erythrocytes in vitro. Scanning electron microscopy revealed that under certain treatments (calcium ionophore A23187, SDS, LPA, and incubation at 50°C), erythrocytes shed EDEVs. The differences in the morphology of erythrocytes subjected to EDEVs-stimulating treatments suggest distinct mechanisms of EDEVs formation. Raman spectroscopy showed that EDEVs obtained by heat treatment may contain hemoglobin, while SDS-induced treatment produces hemoglobin-free vesicles. The data obtained will allow for a targeted selection of EDEVs production methods based on their required composition.

Intercellular communication is a critical component of maintaining tissue homeostasis. One of the communication methods is the transfer of a wide range of biologically active molecules – proteins, nucleic acids (primarily non-coding RNA) and lipids – in extracellular vesicles (EV). This allows EV-producing cells to change the metabolic and transcriptional activity of individual target cell populations over a wide range and “tune” it in accordance with tissue needs. Most of the existing studies are focused on the composition and functions of EVs secreted into the external environment, while the study of the membrane-associated vesicle subclass (membrane-associated vesicles, MAV) is hampered by the lack of generally accepted methods for their isolation from different cell types, including cultured ones, and characterization. In this paper, we present a protocol for isolating the MAV fraction of cultured mesenchymal stromal/stem cells while preserving the morphology and viability of the cells themselves using hyaluronidase treatment. This protocol demonstrated the highest efficiency compared to other tested methods and allows one to obtain a fraction significantly enriched in vesicles, as demonstrated by nanoparticle tracking analysis and transmission electron microscopy. A comparative analysis of the characteristics of MAV and EV secreted into the medium showed that the proposed MAV isolation protocol allows one to obtain a fraction with a similar particle concentration, but they are smaller in size compared to EV. Thus, the isolation method we described allows one to obtain a MAV fraction for further analysis of their composition and functional features.

In this study the computer program Veronica was created – a specialized environment for marking images obtained by cryo-electron microscopy. It allows to highlight the contours of closed and unclosed objects and save them in a convenient format for analysis, as well as to determine the specified metrics for the study of morphology. The Veronica program was used to characterize the morphology of samples of small extracellular vesicles isolated from gastric juice of patients with gastric adenocarcinoma and conditionally healthy donors. An approach to image partitioning and calculation of indices to describe vesicles based on the measurement of nesting (a measure of the number of particles nested within each other) and branching (a measure of the number of adjacent particles surrounded by a common membrane) parameters has been proposed.

The intermediate-conductance calcium-activated potassium channel K_Ca3.1 promotes calciumdependent hyperpolarization of the cell membrane. Its malfunction has been observed in autoimmune and oncological diseases. To study this channel and its peptide blockers using fluorescence analysis, plasmids encoding the α-subunit K_Ca3.1 fused with the fluorescent protein mKate2 at the N- or C-terminus were constructed, and the fluorescent ligand ChTx-GFP was obtained, which is a combination of the peptide blocker charybdotoxin and the green fluorescent protein. It was found that mKate2 at the N-terminus of the α-subunit blocks the transport of the channel into the plasma membrane of Neuro-2a cells, while mKate2 at its C-terminus does not interfere with the efficient accumulation of the channel in the plasma membrane and the formation of a regular tetrameric structure capable of binding peptide blockers. The ligand ChTx-GFP binds to the K_Ca3.1 channel on the membrane at a concentration of 20 nM and can be used for fluorescent imaging of these channels in mammalian cells.

Genetic material of the cell in interphase nucleus is present in a form of a dense DNA-protein structure named chromatin. Structure and dynamics of single nucleosome, which is the basic unit of DNA compactization, is currently well-studied, although the data about the structural and functional organization of higher-level chromatin folding, is still scarce. In the present work, a method of visualization of polynucleosomal constructs using atomic force microscopy is proposed. Polynucleosome assembly on a plasmid with the use of recombinant histone octamers was demonstrated. It was established that glutaraldehyde treatment of polynucleosome sample before its immobilization on a surface preserves nucleosomes, and their height and width corresponds well with previously obtained data. Plasmids themselves were predominantly In extended conformation, which may help studying DNA-protein interactions.

Transcription in the cell is carried out by specialized enzymes – RNA polymerases. RNA polymerases transcribe DNA with the formation of elongation complexes (EC), which have a regulatory significance. Cryo-electron microscopy (cryo-EM) allows obtaining the structures of these complexes and clarifying the mechanisms of transcription stages. However, the preparation of EC samples suitable for cryo-EM studies presents certain challenges. In this work, a selection of protocols for the preparation of EC+39 samples was carried out. The formation of complexes is confirmed by the results of electrophoresis and negative stain electron microscopy. The results obtained can be used to study EC+39 by the cryo-EM method.

Chromatin of eukaryotic organisms is a complexly organized and dynamic complex. Chromatin proteins provide proper regulation of gene expression, DNA replication, and DNA repair. Among the most important regulators of chromatin architecture among non-histone proteins are p53 and PARP1, which are involved in the cellular response to DNA damage. In the present study, we investigated the cooperative and competitive binding of the DNA-binding domain (DBD) of p53 and the enzyme PARP1 to mononucleosomes reconstituted on the Widom 603 sequence with an embedded p53 binding site. To detect interactions, the electrophoretic mobility shift assay (EMSA) method with fluorescently labeled DNA. Complexes were formed in two ways: nucleosomes were pre-incubated with p53 DBD and then PARP1 was added, or the nucleosome–PARP1 complex was formed first and then p53 DBD was introduced. The results showed that the order of protein addition determines the nature of their interaction with the nucleosome: at low p53 concentrations, displacement of this protein by PARP1 is observed, while at higher p53 concentrations, stable nucleosome–p53 complexes are formed, undisturbed by PARP1. No stable ternary nucleosome–p53–PARP1 complexes were detected.

This study establishes a real-time fluorescence microscopy platform for visualizing ligand binding dynamics to His-tagged proteins bound to Ni-NTA agarose beads. By preserving solution-phase kinetics while enabling sub-minute temporal resolution in physiological buffers, the methodology overcomes critical limitations of surface-based techniques and gel electrophoretic methods. We applied this platform to investigate inhibitor action within a nucleosomal system, a more physiologically relevant context than free DNA. Through studies of PARP2-nucleosome interactions modulated by clinical inhibitors (talazoparib, veliparib) and by reaction of poly(ADP-ribosyl)ation in the presence of NAD+, we demonstrate direct spatial and temporal resolution of chromatin-protein dynamics. The platform’s virtually unlimited buffer compatibility, real-time monitoring capabilities, and elimination of covalent immobilization artifacts provide transformative insights into drug mechanisms and chromatin engagement processes.

PARP3 (Poly(ADP-ribose) polymerase 3), like other PARP family members (PARP1 and PARP2), is an important factor in DNA repair. The specific functions and molecular mechanisms of action of this protein remain insufficiently studied. The development of a reliable protocol for obtaining high-purity and yield PARP3 is essential for comprehensive protein analysis, enzymatic activity, including studies of its binding to DNA, interactions with other protein factors, and structural investigations. In this study, we present a modified protocol for the expression of human PARP3 from Escherichia coli cells and its subsequent purification, which significantly increases protein yield compared to previously published methods.

Carotenoid-protein complexes play a crucial role in photosynthesis, photoreception, protection against oxidative stress, metabolism, and pigmentation. This study conducts a detailed analysis of structural data with atomic resolution for carotenoid-containing proteins. The research examines molecular features of carotenoid-binding regions and structural characteristics of bound carotenoids. The findings reveal general principles of the protein-carotenoid interface organization, essential for developing new approaches to targeted modification. Additionally, a machine learning model is created to predict carotenoid-binding activity based on the primary protein structure.

Translation pre-initiation complexes are typical components of the cytoplasm of eukaryotic cells, consisting of 40S ribosomal subunits bound to initiation factors. Actively studied in mammals and protozoa, these complexes are involved in the initiation of mRNA translation. We studied the abundance and structure of plant pre-initiation complexes using single particle cryo EM analysis of wheat germ extract preparations. It was found that about 29% of free 40S subunits form a complex with the initiation factors eIF3 and eIF1A. The structure of the complex was reconstructed with overall resolution better than 3 Å and the core of factor eIF3 with a resolution of 3.6 Å. These are the first structural data on the structure of initiation ribosomal complex in plants. We constructed the atomic models of the iIF3 core and distal subunits which revealed notable differences from those in mammalian cells.

Using cryo-electron microscopy, we studied the structure of the axial fiber protein gp56, a component of the adsorption apparatus of the Stx-converting phage phi24B. The axial fiber is highly mobile, and the gp56 trimer is encased by the bulky hexameric nozzle protein gp57, creating a symmetry-mismatch interface that complicates three-dimensional reconstruction. By applying symmetry expansion and local orientation refinement, we generated a density map of the axial fiber, visualized the tertiary structure of its globular domains, and determined their positions relative to the other proteins of the phi24B adsorption apparatus.

One of the traditional approaches to the creation of live attenuated vaccines is cold adaptation of the virus to produce temperature-sensitive (ts) mutants. In this work, we investigated the mor phological features and antigenic properties of the attenuated ts mutant F-F3 SARS-CoV-2 in comparison with the parent strain FEB2 (Omicron BA.5.2). Transmission electron microscopy of the virus inactivated by ultraviolet radiation revealed no significant differences in the morphol ogy of negatively contrasted virus particles and S-spikes: a characteristic “crown” consisting of spikes in the native prefusion conformation was found around the virions. Cryoelectron micros copy of the parent strain confirmed the presence of S-spikes in the prefusion conformation, while the ts mutant was not studied by this method due to insufficient virion concentration. In cubation with immune sera against an omicron-like strain revealed the formation of immune complexes both in the case of the parent strain and the ts mutant. Atomic force microscopy sug gested the presence of single virions in the preparation, but did not reveal a characteristic corona around them. This may be due to the fragility of S-spikes destroyed during sample preparation, or to the masking effect of serum albumin aggregates from the culture cell medium.

The histone chaperone FACT plays a key role in chromatin reorganization by mediating ATP-in dependent nucleosome unwinding. The yeast yFACT complex consists of the Spt16 and Pob3 subunits, which form a heterodimer functionally associated with the non-histone protein Nhp6. In this study, negative-stain transmission electron microscopy was used to investigate the inter action of the yFACT complex containing the Pob3 subunit with the C-terminal domain (CTD) removed with the nucleosome in the presence of Nhp6. As a result of CTD removal, the efficien cy of FACT binding to the nucleosome decreased by a factor of 2, and the ability to fully unfold the nucleosome was impaired: instead of the almost symmetrical, fully unfolded structures char acteristic of wild type yFACT, asymmetrical, partially unfolded structures were observed. The data obtained indicate the key role of the Pob3 CTD in ensuring FACT binding to the nucle osome, which is important for understanding the mechanisms of chromatin remodeling and transcription regulation.

Cryo-ET (cryo-electron tomography) is a powerful tool for studying the structure of biological objects in their native state. However, Cryo-ET is still not widely used to study cellular organelles including cell nucleus and chromatin in situ. In our work, we aimed to investigate the possibilities of employing Cryo-ET for studying the architecture of natively preserved chromatin, focusing on the possibility of using a cryoCLEM (cryo-correlative light and electron microscopy) approach to target final steps of sample preparation by focused ion beam (FIB) milling exactly to the area of interest – specifically, heterochromatin loci.