The IV Russian International Conference on Cryo-Electron Microscopy (RICCEM-2023) was held at the Faculty of Biology of Lomonosov Moscow State University in a hybrid (offline and online) format and brought together more than 250 researchers from 10 countries. Following the conference, a special issue of the journal “Lomonosov Biology Journal” (“Vestnik Moskovskogo universiteta. Seria 16. Biologia”) was published. This article briefly summarizes the main results of the conference and the content of the works published in the special issue.

The current state of the problem of folding proteins and other biopolymers is discussed. The concept of a multidimensional potential energy surface and a free energy surface for linear polymers is detailed, taking into account the topology of the configuration space and the presence of symmetry elements with respect to the permutation of identical monomer units. The presence of kinematic bonds for conformational movements in a viscous medium leads to a tendency to form helical structures of linear polymers. The dynamic effects of viscosity also lead to an almost uniform distribution of energy dissipation rates over the chain nodes. The combination of potential energy surface topography and viscosity effects provides a physical basis for the development of the theory of folding in the direction of interpreting various experimental observations and elucidating the principles for the formation of an amino acid code for 3D protein structures. The relationship between the denaturation temperature of the folded state of the biopolymer and the energy of non-valent interactions between monomers in the chain is analyzed.

Wandering spiders (family Ctenidae) have multicomponent venoms in which more than 500 different peptides and proteins, called ctenitoxins, have been identified. The main components of the venom are cysteine-rich peptides containing an inhibitory cystine knot (ICK) motif. The pharmacological diversity of ctenitoxins allows us to consider some of them as prototypes for the development of new drugs for the treatment of chronic pain, Huntington’s disease, erectile dysfunction and glaucoma. According to the location of cysteine residues in the amino acid sequence, ctenitoxins are divided into 14 groups containing from 6 to 14 Cys residues. Currently, the spatial structure of only one ctenitoxin, ω-CNTX-Pn4a (Pha1β or Tx3-6) from the Brazilian wandering spider Phoneutria nigriventer, has been determined. Another 10 structural groups of ctenitoxins have homology with the known spatial structures of spider toxins of other families and other proteins, and for three groups the structural homologues are unknown. In this paper, we proposed possible disulfide bonding patterns for all groups of ctenitoxins. A comparison of the obtained schemes with the predictions of the AlphaFold 2.0 program shows that this neural network does not always correctly predict the structures of cysteine-rich peptides, especially if the structures of mature molecules without leader sequences are modeled.

Cryo-electron microscopy (cryo-EM) is indispensable for the structural studies of enveloped viruses – dangerous pathogens of humans and animals. Yet, it requires highly specialized equipment as well as careful sample preparation. In this work, the capabilities of transmission electron microscope JEOL JEM-2100 equipped with cryo-transfer holder are used, and preliminary cryo-EM data for influenza A and B virus strains and SARS-CoV-2 inactivated with beta-propiolactone are presented. Image analysis allows: (1) to distinguish “empty” viral particles from “full” ones (containing nucleocapsid); (2) to visualize the lipid bilayer of the viral envelope; (3) identify influenza virus surface antigens and the M1 protein layer combined with the inner lipid monolayer; (4) distinguish different morphology of S-spikes on the surface of inactivated SARS-CoV-2 virions. The developed approach provides good image quality for both fundamental and applied research.

A detergent-free protocol for purification of the coronavirus prefusion S-protein using styrenemaleic acid copolymer (SMA) was developed. Expression of the S-protein was carried out in HEK293T cells. Two solubilization methods were used to purify and prepare the S-protein for microscopy: in NP-40 detergent and as part of SMA. The resulting preparations were examined in an electron microscope, and the particles of purified S-proteins were classified. Analysis of two-dimensional projections of the particles showed that the use of lipodiscs for solubilization leads to lower mobility of the purified protein on the substrate, compared to the protein in the detergent, which may further contribute to obtaining higher resolutions when studying the structure of membrane proteins.

The study involved the fabrication of films with different roughness and scaffolds made of poly(3-hydroxybutyrate) using various methods. Chaotic and oriented scaffolds with varying fiber thickness were obtained through the electrospinning method, depending on the polymer concentration and electrospinning parameters. Films with different surface roughness were obtained using spin coating and self-assembly methods. It was demonstrated that the varying microstructure of the surface does not affect the growth of mesenchymal stem cells over the course of a week; however, it does influence the morphology of the adhered cells.

In this work, conditions were selected for obtaining a sample of eukaryotic chaperonin TRiC suitable for studying by cryo-electron microscopy. Using the method of differential scanning (time-resolved) fluorimetry, the temperature stability of protein samples at different concentrations of salt and glycerol was compared, and then the selected conditions were used to prepare the sample for microscopy. As a result, the structure of bovine TRiC in an open conformation was obtained at 4.42 Å resolution.

Candida auris is a pathogenic fungus that causes infections in people with weakened immune systems. It is resistant to antimicrobial drugs, which complicates treatment. C. auris poses a serious threat to public health, as it has a high resistance and contagiousness. The ribosome plays an important role in the survival and development of this infection, and the search for inhibitors of the C. auris ribosome is of great importance. We used cryoelectron microscopy and single particle analysis to obtain the three-dimensional structure of the C. auris ribosome. We describe the architecture of the ribosome subunits and their interactions, providing valuable information for the development of novel anti-fungals against C. auris.

This paper demonstrates an example of a successful upgrade of a JEOL JEM-2100 analytical transmission electron microscope to a low-resolution cryo-electron microscope designed for routine tasks of sample preparation and quality evaluation. As a result of the upgrade, the instrument allows the subnanometer resolution of protein molecule reconstructions (within 8 Å). The influence of graphene and amorphous carbon support films to prevent the effect of preferred orientation of protein particles in the frozen sample is discussed.

This paper is a technical and methodological note, the purpose of which is to introduce into the practice of biological research methods of cryomicroscopy in a conveyor mode, starting from small magnifications and ending with the limits of magnification/resolution of scanning electron cryomicroscopy. The protocol described can be applied to the samples with low sample preparation complexity without ultratomy or the sample processing typical for transmission electron microscopy methods. According to this protocol samples are analyzed in a single microcuvette (chip) indexed by laboratory information management system and sequentially moved from the non-destructive low-resolution optical microscopy instruments (such as lensless cryomicroscopes) and optical super-resolution methods (some microinterferometers and MIMs with cryotables) to the CryoSEM/CryoESEM level (in programmable environments and atmospheres). Methods of correlation lensless cryomicroscopy and scanning microscopy (including those with the subsequent transition to microanalysis) were introduced; CryoCUVEM and CryoCIREM methods in the ultraviolet and infrared range, respectively; microinterferometry methods using a multi-beam reflected light interferometer (based on the MII-11 platform with several changes); the development of CryoCDICEM systems based on the optical path of an inverted metallographic microscope with a DIC attachment and a LED emitter was also initiated. The advantages of cryoconveyor analysis protocols are ensuring the sample safety in a single cuvette-chip and the possibility of establishing spatial colocalization between the data of optical and electron microscopy (including in the CLEM/CryoCLEM mode), as well as providing a comprehensive nondestructive sample analysis in the sequential study of the microscopic systems with the possibility of varying the subsequent stages of high-resolution microscopy, depending on the results obtained at the previous stages of lower resolution microscopic studies.

The rheological properties of hydrogels of a natural polysaccharide sodium alginate and small amount of clay nanotubes of halloysite were investigated. Changes of rheological properties during the transition from a semi-dilute polymer solution to a hydrogel upon cross-linking by calcium ions were shown. In the gel state, the samples have a yield stress, and their viscosity decreases with the shear rate, but the properties are quickly recovered after the load removal. It was obtained that the addition of up to 0.3 vol.% nanotubes of natural clay halloysite leads to an increase by several times of a storage modulus and an yield stress of the hydrogels. At the same time, the practically important properties of shear thinning and the rapid recovery of properties after the load removing make the nanocomposite hydrogels of alginate and halloysite nanotubes promising for use as ink for extrusion 3D printing.