Biology subjects

Kudryashev, M.

Publications and source records attributed to Kudryashev, M..

12 recordsLinked to original sources

bioRxiv2025

Cryo-EM structure of the naked mole-rat ribosome reveals a stabilized split 28S rRNA

The naked mole-rat (Heterocephalus glaber) is a long-lived mammal with remarkable resistance to cancer and hypoxia, suggesting the evolution of robust proteostasis networks. The ribosome, the central engine of protein synthesis, is key for cellular stress responses and has an unusual feature of the 28S rRNA split; however, the details of its organization remain unknown. Here, we present high-resolution cryo-EM structures of the naked mole-rat 80S ribosome in two states of the elongation cycle. The structures reveal a conserved overall architecture and rRNA modification landscape compared to other mammals, and provide an atomic-level view of the unique break in the 28S rRNA. This cleavage event, located in the D6 expansion segment, is structurally stabilized by a network of interactions with surrounding ribosomal proteins, maintaining the integrity of the large subunit. Our comparative analysis revealed that this compensatory network preserves a canonical architecture nearly indistinguishable from intact mouse and human ribosomes. These findings resolve the structural basis of this unique cleavage, showing that it is a stable, integrated feature whose function is likely linked to more subtle regulatory mechanisms, rather than inducing major structural rearrangements.

biochemistry↗

bioRxiv2025

Integrative structural interactomics reveals protein organization and structure in a giant virus

Giant viruses are large DNA viruses that infect unicellular and multicellular eukaryotes and form exceptionally large extracellular particles. (Meta)genomics and (meta)transcriptomics have provided insight into their diverse coding repertoire, but many of the proteins remain to be characterized as they lack homology with known proteins. Here, we integrated cross-linking mass spectrometry, quantitative proteomics, computational tools and cryo-EM data to characterize the protein architecture of intact melbournevirus particles. Based on this, we allocated 88 viral proteins to different virion sub-compartments and proposed topologies of 25 inner membrane proteins. We assigned eight components of the capsid in cryo-EM data, including proteins that tether the capsid shell to the membrane, reflecting key points in virion maturation. The data provide a valuable resource and demonstrate the power of an integrative approach to gain system-level structural insights into a poorly characterized biological system.

biochemistry↗

bioRxiv2025

Structures of EHD2 filaments on curved membranes provide a model for caveolar neck stabilization

Caveolae are flask-shaped invaginations of the plasma membrane serving critical functions in mechano-protection and signal transduction. Caveolar dynamics, such as movement within the plasma membrane or endocytosis, relies on precise shaping of the highly curved caveolar necks. The dynamin-like EHD2 ATPase is proposed to form an oligomeric scaffold around the caveolar neck, but its detailed molecular action is poorly understood. Here, we employed cryo-electron tomography to elucidate structures of EHD2 filaments oligomerized on tubulated liposomes. EHD2 filaments form a highly curved membrane scaffold which stabilizes a sophisticated tubular membrane geometry with undulations along the tubes axis. An amino-terminal sequence facilitates this geometry by inserting into the membrane, thereby acting as a spacer between adjacent filaments. Moreover, in endothelial cells lacking EHD2, caveolar necks become narrower and elongated. Our structural work provides the molecular framework for understanding EHD2 scaffold formation and its cellular function in caveolar dynamics.

molecular biology↗

bioRxiv2025

Dynamic nanoscale architecture of synaptic vesicle fusion in mouse hippocampal neurons

During neurotransmission, presynaptic action potentials trigger synaptic vesicle fusion with the plasma membrane within milliseconds. To visualize membrane dynamics before, during, and right after vesicle fusion at central synapses under near-native conditions, we developed an experimental strategy for time-resolved in situ cryo-electron tomography with millisecond temporal resolution. We coupled optogenetic stimulation with cryofixation and confirmed the stimulation-induced release of neurotransmitters via cryo-confocal microscopy of a fluorescent glutamate sensor. Our morphometric analysis of tomograms from stimulated and control synapses allowed us to characterize five states of vesicle fusion intermediates ranging from stalk formation to the formation, opening, and collapsing of a fusion pore. Based on these measurements, we generated a coarse-grained simulation of a synaptic vesicle approaching the active zone membrane. Both, our morphofunctional and computational analyses, support a model in which calcium-triggered fusion is initiated from synaptic vesicles in close proximity to the active zone membrane, whereby neither tight docking nor an induction of membrane curvature at the active zone are favorable. Numbers of filamentous tethers closely correlated to the distance between vesicle and membrane, but not to their respective fusion readiness, indicating that the formation of multiple tethers is required for synaptic vesicle recruitment preceding fusion.

neuroscience↗

bioRxiv2024

Denoising, Deblurring, and optical Deconvolution for cryo-ET and light microscopy with a physics-informed deep neural network DeBCR

Computational image-quality enhancement for microscopy (deblurring, denoising, and optical deconvolution) provides researchers with detailed information on samples. Recent general-purpose deep learning solutions advanced in this task. Yet, without consideration of the underlying physics, they may yield unrealistic and non-existent details and distortions during image restoration, requiring domain expertise to discern true features from artifacts. Furthermore, the large expressive capacity of general-purpose deep learning models requires more resources to train and use in applications. We introduce DeBCR, a physics-informed deep learning model based on wavelet theory to enhance microscopy images. DeBCR is a light model with a fast runtime and without hallucinations. We evaluated the image restoration performance of DeBCR and 12 current state-of-the-art models over 6 datasets spanning crucial modalities in advanced light microscopy and cryo-electron tomography. Leveraging optic models, DeBCR demonstrates superior performance in denoising, optical deconvolution, and deblurring tasks across both LM and cryo-ET modalities.

bioinformatics↗

bioRxiv2024

Molecular architecture of synaptic vesicles.

Synaptic vesicles (SVs) store and transport neurotransmitters to the presynaptic active zone for release by exocytosis. After release, SV proteins and excess membrane are recycled via endocytosis, and new SVs are formed in a clathrin-dependent manner. This process maintains the morphology and complex molecular composition of SVs through multiple recycling rounds. Previous studies explored the molecular composition of SVs through proteomic analysis and fluorescent microscopy, proposing a model for an average SV1,2. However, the structural heterogeneity and molecular architecture of individual SVs are not well described. Here we used cryo-electron tomography to visualize morphological and molecular details of SVs isolated from mouse brains and inside cultured neurons. We describe several classes of small proteins on the SV surface and long proteinaceous densities inside SVs. We identified V-ATPases, determined a structure using subtomogram average, and showed them forming a complex with the membrane-embedded protein synaptophysin. Our bioluminescence assay revealed pairwise interactions between VAMP2 and synaptophysin and V-ATPase Voe1 domains. Interestingly, V-ATPases were randomly distributed on the surface of SVs irrespective of vesicle sizes. A subpopulation of isolated vesicles and vesicles inside neurons contained a partially assembled clathrin coat with a soccer-ball symmetry. We observed a V-ATPase under clathrin cage in several isolated clathrin-coated vesicles. Additionally, from isolated SV preparations and within hippocampal neurons we identified clathrin baskets without vesicles. We determined their preferential location in proximity to the cell membrane. Our analysis advances the understanding of individual SVs diversity and their molecular architecture.

biophysics↗

bioRxiv2023

Elucidating the activation mechanism for GBP1 oligomerization

The dynamin-related human guanylate-binding protein 1 (GBP1) mediates host defense against microbial pathogens. Upon GTP binding and hydrolysis, auto-inhibited GBP1 monomers dimerize and assemble into soluble and membrane-bound oligomers, which are crucial for innate immune responses. How higher-order GBP1 oligomers are built from dimers and how assembly is coordinated with nucleotide-dependent conformational changes has remained elusive. Here, we present cryo-electron microscopy-based structural data of the soluble and membrane-bound GBP1 oligomers demonstrating that GBP1 assembles in an outstretched dimeric conformation. We identify a surface-exposed helix in the large GTPase domain, which contributes to the oligomerization interface, and probe its nucleotide- and dimerization-dependent movements facilitating the formation of an antimicrobial protein coat on a Gram-negative bacterial pathogen. Our results reveal a sophisticated activation mechanism for GBP1 in which nucleotide-dependent structural changes coordinate dimerization, oligomerization, and membrane binding to allow encapsulation of pathogens with an antimicrobial protein coat.

biochemistry↗

bioRxiv2023

Determining The Structure of the Bacterial Voltage-gated Sodium Channel NaChBac Embedded in Liposomes by Cryo Electron Tomography and Subtomogram Averaging

Voltage-gated sodium channels shape action potentials that propagate signals along cells. When the membrane potential reaches a certain threshold, the channels open and allow sodium ions to flow through the membrane depolarizing it, followed by the deactivation of the channels. Opening and closing of the channels is important for cellular signalling and regulates various physiological processes in muscles, heart and brain. Mechanistic insights into the voltage-gated channels are difficult to achieve as the proteins are typically extracted from membranes for structural analysis which results in the loss of the transmembrane potential. Here, we report the structural analysis of a bacterial voltage-gated sodium channel, NaChBac, reconstituted in liposomes under an electrochemical gradient by cryo electron tomography and subtomogram averaging. We show that the small channel, most of the residues of which are embedded in a membrane, can be localized using a genetically fused GFP. GFP can aid the initial alignment to an average resulting in a correct structure, but does not help for the final refinement. At a moderate resolution of [~]16 [A] the structure of NaChBac in an unrestricted membrane bilayer is 10% wider than the structure of a purified protein previously solved in nanodiscs, suggesting the potential movement of the peripheral voltage-sensing domains. Our structural analysis explores the limits of structural analysis of membrane proteins in membranes. HIGHLIGHTSO_LIStructural analysis of the bacterial voltage-gated sodium channel NaChBac in lipid vesicles under the resting membrane potential by cryo electron tomography and subtomogram averaging. C_LIO_LIFused GFP allows identification of a 120-kDa mostly transmembrane protein in tomograms, and helps for the initial alignment but not for the final refinements. C_LIO_LIThe map of NaChBac in liposomes at a resolution of 16.3 [A] is [~]10% wider than the protein structure in a nanodisc. C_LI

biochemistry↗

bioRxiv2023

Streamlined Structure Determination by Cryo-Electron Tomography and Subtomogram Averaging using TomoBEAR

Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to solve such structures in situ at resolutions in the range of 3 Angstrom for some macromolecules. In order to be applicable to structural determination of the majority of macromolecules observable in cells in limited amounts, processing of tomographic data has to be performed in a high-throughput manner. Here we present TomoBEAR - a modular configurable workflow engine for streamlined processing of cryo-electron tomographic data for subtomogram averaging. TomoBEAR combines commonly used cryo-EM packages and reasonable presets to provide a transparent "white box" for data management and processing. We demonstrate applications of TomoBEAR to two datasets of purified proteins and to a membrane protein RyR1 in a membrane and demonstrate the ability to produce high resolution with minimal human intervention. TomoBEAR is an open-source and extendable package, it will accelerate the adoption of in situ structural biology by cryo-ET.

biophysics↗

bioRxiv2021

KAHRP dynamically relocalizes to remodeled actin junctions and associates with knob spirals in P. falciparum-infected erythrocytes

The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite-encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually co-localizes with remnant actin junctions. We further present a 35[A] map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected [~]60 KAHRP molecules per knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold.

microbiology↗

bioRxiv2020

Subnanometer-resolution structure determination in situ by a hybrid subtomogram averaging - single particle cryoEM - workflow

Cryo electron tomography (cryo-ET) combined with subtomogram averaging (StA) enables structural determination of macromolecules in their native context. A few structures were reported by StA at resolution higher than 4.5 [A], however all of these are from viral structural proteins or vesicle coats. Reaching high resolution for a broader range of samples is uncommon due to beam-induced sample drift, poor signal-to-noise ratio (SNR) of images, challenges in CTF correction, limited number of particles. Here we propose a strategy to address these issues, which consists of a tomographic data collection scheme and a processing workflow. Tilt series are collected with higher electron dose at zero-degree tilt in order to increase SNR. Next, after performing StA conventionally, we extract 2D projections of the particles of interest from the higher SNR images and use the single particle analysis tools to refine the particle alignment and generate a reconstruction. We benchmarked our proposed hybrid StA (hStA) workflow and improved the resolution for tobacco mosaic virus from 7.2 to 5.2 [A] and the resolution for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 [A]. We demonstrate that hStA can improve the resolution obtained by conventional StA and promises to be a useful tool for StA projects aiming at subnanometer resolution or higher.

biochemistry↗

bioRxiv2018

Inner-membrane GspF of the bacterial type II secretion system is a dimeric adaptor mediating pseudopilus biogenesis

The type II secretion system (T2SS), a protein complex spanning the bacterial envelope, is pivotal to bacterial pathogenicity. Central to T2SS function is the extrusion of protein cargos from the periplasm into the extracellular environment mediated by a pseudopilus and motorized by a cytosolic ATPase. GspF, an inner-membrane component of T2SS has long been considered to be a key player in this process, yet the structural basis of its role had remained elusive. Here, we employed single-particle electron microscopy based on XcpS (GspF) from the T2SS of pathogenic P. aeruginosa stabilized by a nanobody, to show that XcpS adopts a dimeric structure mediated by its transmembrane helices. This assembly matches in terms of overall organization and dimensions the basal inner-membrane cassette of a T2SS machinery. Thus, GspF is poised to serve as an adaptor involved in the mediation of propeller-like torque generated by the motor ATPase to the secretion pseudopilus.\n\nNon-technical author summaryAntibiotic resistance by bacteria imposes a worldwide threat that can only be overcome through a multi-front approach: preventive actions and the parallel development of novel molecular strategies to combat antibiotic resistance mechanisms. One such strategy might focus on antivirulence drugs that prevent host invasion and spreading by pathogenic bacteria, without shutting down essential functions related to bacterial survival. The rationale behind such an approach is that it might limit selective pressure leading to slower evolutionary rates of resistant bacterial strains. Bacterial secretion systems are an appropriate target for such therapeutic approaches as their impairment will inhibit the secretion of a multitude of virulence factors. This study focuses on the structural characterization of one of the proteins residing in the inner-membrane cassette of the type II secretion system (T2SS), a multi-protein complex in multiple opportunistic pathogens that secretes virulence factors. The targeted protein is essential for the assembly of the pseudopilus, a rod-like supramolecular structure that propels the secretion of virulence factors by pathogenic Gram-negative bacteria. Our study crucially complements growing evidence supporting a rotational assembly model of the pseudopilus and contributes to a better understanding of the functioning of the T2SS and the related secretion systems. We envisage that such knowledge will facilitate targeting of these systems for therapeutic purposes.

microbiology↗