Biology subjects

Novacek, J.

Publications and source records attributed to Novacek, J..

16 recordsLinked to original sources

bioRxiv2026

Open or closed? Exploring the conformational heterogeneity of human plasminogen across multiple resolution scales

Plasminogen (Plg), the zymogen of the fibrinolytic protease plasmin, is a multidomain, conformationally-rich protein that plays a crucial role in targeted thrombolysis for stroke and heart attack treatments. Yet, the conformational state that affects binding to Plg activators and fibrinolytic activity is rarely validated, contributing to poor reproducibility and failures in drug development. Here, we establish a structural biology workflow to characterize human Plg suspended in an aqueous solution, preserving it in a near-native state and eliminating the need for crystal growth. Two variants, cleavage-resistant Plg-RV (R561A) and catalytically inactive Plg-CAT (S741A), were expressed in mammalian cells. Both variants were correctly folded and stable (Tm {approx} 60.6{degrees}C) and, unlike commercial plasma-derived Plg, were resistant to staphylokinase-mediated activation. Small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry revealed substantial conformational heterogeneity. The recombinantly-produced Plg variants adopted a closed conformation, exhibiting a good fit to the closed structure determined by X-ray diffraction. Conversely, the plasma-derived Plg populated an extended, open-like state. Cryo-EM analysis of the Plg-RV variant yielded a 4.4 [A] resolution map, and a rigid-body-fitted model revealed the closed-state architecture. Our findings demonstrate that rigorous structural validation of Plg is essential for future functional studies and rational development of next-generation thrombolytic agents.

biochemistry↗

bioRxiv2026

Automated cryo-volume EM for high-resolution 3D imaging and in situ structural analysis of cells and tissues

Cryo-volume electron microscopy (CVEM) enables three-dimensional imaging of biological ultrastructure in a near-native state but has been limited by low image contrast and charging artifacts that hinder data interpretation and complicate automation of data acquisition. Here we present an experimental and computational workflow that combines orthogonal cryo-SEM imaging, spot-geometry optimized O+ plasma-FIB milling, dedicated acquisition-control routines, and dedicated image alignment procedure. The workflow enables autonomous acquisition of volumetric datasets from vitrified cells and tissues at [~]15-20 nm isotropic resolution. In addition, sub-volume averaging of 113 nuclear pore complexes extracted from CVEM dataset of Cos-7 cell yielded its reconstruction at 9.4 nm resolution. Together, these results establish CVEM as a robust platform for autonomous high-resolution volumetric imaging and structural analysis of vitrified biological specimens.

Cell Biology↗

bioRxiv2025

Antagonistic insulin mimetics lock the insulin receptor in an alternative apo-state

Despite significant advancements in high-resolution structural analysis of activated human insulin receptor (IR), the molecular mechanisms underlying its conformational plasticity that govern the transition from the apo state to the activated state are still not well understood. This leaves critical aspects of IR regulation unclear. Here, we reveal the mechanism by which the insulin mimetics Ada, Trim, and S661 fully inhibit the insulin receptor. The receptor is stabilized in a yet structurally un-described {cap}-shaped conformation which is induced by antagonist binding between the L1 and FnIII-1 domains. In contrast to insulin-bound IR structures, the -CT helix is not observable in the {cap} conformation, and the membrane-proximal regions of the FnIII-3 domains are >10 nm apart, which prohibits transmembrane signal transduction and kinase domain activation. Analysis of apo-IR electron cryo-microscopy data indicates that the {cap}-shaped state is one of several metastable apo-IR conformations. These findings underscore the intrinsic conformational dynamics of apo-IR and its role in integrating insulin binding and receptor activation.

molecular biology↗

bioRxiv2025

Mechanisms of transcription attenuation and condensation of RNA polymerase II by RECQ5

The elongation rates of RNA polymerase II (RNAPII) require precise control to prevent transcriptional stress, which can impede co-transcriptional pre-mRNA processing and contribute to many age- or disease-associated molecular changes (e.g., loss of proteostasis)1-5. Additionally mesoscale organization of transcription is thought to control the transcriptional rates6 and multiple factors have been reported to form biomolecular condensates and integrate RNAPII through the interaction with the C-terminal domain (CTD) of the largest subunit, RPB17-10. However, the structural organization of these condensates remains uncharacterized due to their small size and inherently dynamic nature. Here, we investigated the molecular mechanisms by which a general transcription factor - RECQ5 - associates with hyperphosphorylated RNAPII elongation complex (P-RNAPII EC) and controls translocation of RNAPII along genes. We combined biochemical reconstitution, electron cryomicroscopy, cryotomography, and coarse-grained simulations. We report two mechanisms by which RECQ5 modulates RNAPII transcription. At the atomic level, we demonstrate that RECQ5 uses the brake helix as a doorstop to control RNAPII translocation along DNA, attenuating transcription. At the mesoscale level, RECQ5 forms a condensate scaffold matrix, integrating P-RNAPII EC through a network of site-specific interactions, reinforcing the condensates structural integrity. Our integrative, multi-scale study provides insights into the structural basis of transcription attenuation and into the molecular architecture and biogenesis of a model RNAPII condensate.

biochemistry↗

bioRxiv2024

Cell attachment and tail contraction of S. aureus phage phi812

Phages with contractile tails employ elaborate mechanisms to penetrate bacterial cell walls and deliver their genomes into the host cytoplasm. Here, we used cryo-EM to show that the baseplate of phage 812, a member of the Kayvirus genus, which infects Gram-positive Staphylococcus strains, is formed of a core, wedge modules, and baseplate arms carrying receptor-binding proteins 1 and 2 and tripod complexes. Upon binding to a host cell, the receptor-binding proteins of phage 812 baseplate reorient and undergo conformational changes. The changes to the tripod complexes trigger the release of the central spike and weld proteins, which expose peptidoglycan-degrading domains of the hub proteins. Changes in the positions of baseplate arms are transmitted through wedge modules to tail sheath initiator proteins. The ring of the tail sheath initiator proteins expands and triggers the contraction of the tail sheath, which shortens to 50% and pushes the tail tube 10-30 nm into the bacterial cytoplasm. Homologous molecular mechanisms are probably shared by phages of the Herelleviridae family with contractile tails to infect Gram-positive bacteria.

microbiology↗

bioRxiv2023

The structure of immature tick-borne encephalitis virus

Tick-borne encephalitis virus (TBEV) is a medically important flavivirus that poses a significant health threat in Europe and Asia. However, the structure of the immature form of TBEV remains unknown. Here, we employed state-of-the-art cryogenic electron microscopy (cryoEM) to determine the structure of the immature TBEV particle. The immature TBEV particle has a diameter of 56 nm and its surface glycoproteins are organised into spikes characteristic of immature flaviviruses. The cryoEM reconstructions of the whole virus and of the individual spike enabled us to build atomic models of the major viral components, the E and prM proteins. The insights obtained from our study provide a foundation for understanding the early stages of TBEV assembly and maturation. The pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in metastable conformation. Destabilisation of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM cleavage, the collapse of E protein ectodomains onto the virion surface concurrent with significant movement of the membrane domains of both E and M, and release of the pr fragment from the particle render the virus mature and infectious. This knowledge contributes to our understanding of the flavivirus life cycle.

microbiology↗

bioRxiv2023

Cryo-electron tomography of enterovirus cell entry and endosome escape

Enveloped viruses deliver their genomes into the cell cytoplasm by membrane fusion; in contrast, membrane penetration by non-enveloped viruses is more diverse and less well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. To initiate infection, most enteroviruses enter cells by endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing rhinovirus 2, echovirus 18, echovirus 30, or enterovirus 71 deform, rupture, and release their content into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of enterovirus particles that released their genomes in endosomes, but did not prevent them from reaching the cytoplasm. Inhibiting N-WASP-mediated post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. We show that the rupture of endosomes also occurs in uninfected cells. In summary, our results indicate that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus genomes and particles into the cytoplasm. Since the studied enteroviruses employ different receptors for cell entry but are all delivered into the cytoplasm by cell-mediated endosome disruption, it is possible that many other enteroviruses utilize endosome rupture to infect cells.

microbiology↗

bioRxiv2023

Structure and replication cycle of a virus infecting climate-modulating alga Emiliania huxleyi

The globally distributed marine alga Emiliania huxleyi produces reflective calcite disks (coccoliths) that increase the albedo of ocean water and thus reduce the heat absorption in the ocean, which cools the Earths climate. The population density of E. huxleyi is restricted by nucleocytoplasmic large DNA viruses, including E. huxleyi virus 201 (EhV-201). Despite the impact of E. huxleyi viruses on the climate, there is limited information about their structure and replication. Here we show that the dsDNA genome inside the EhV-201 virion is protected by an inner membrane, capsid, and outer membrane decorated with numerous transmembrane proteins. The virions are prone to deformation, and parts of their capsids deviate from the icosahedral arrangement. EhV-201 virions infect E. huxleyi by using their fivefold vertex to bind to a host cell and fuse the viruss inner membrane with the plasma membrane. Whereas the replication of EhV-201 probably occurs in the nucleus, virions assemble in the cytoplasm at the surface of endoplasmic reticulum-derived membrane segments. Genome packaging initiates synchronously with the capsid assembly and completes through an aperture in the forming capsid. Upon the completion of genome packaging, the capsids change conformation, which enables them to acquire an outer membrane by budding into intracellular vesicles. EhV-201 infection induces a loss of surface protective layers from E. huxleyi cells, which allows the continuous release of virions by exocytosis. Our results provide insight into how EhVs bypass the surface protective layers of E. huxleyi and exploit the organelles of an infected cell for progeny assembly.

molecular biology↗

bioRxiv2023

Ultrastructural details of resistance factors of the bacterial spore revealed by in situ cryo-electron tomography

The bacterial spore owes its incredible resistance capacities to various molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography (cryo-ET) on bacteria lamellae generated by cryo-focused ion beam micromachining (cryo-FIBM) provides insights into the ultrastructural organization of Bacillus subtilis sporangia, including that of the DNA and nascent coat layers. Analysis of the reconstructed tomograms reveal that rather early during sporulation, the chromosome in the developing spore (the forespore) adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a complex stack of amorphous or structured layers with distinct electron density, dimensions and organization. We investigated the nature of the nascent coat layers in various mutant strains using cryo-FIBM/ET and transmission electron microscopy on resin sections of freeze-substituted bacteria. Combining these two cellular electron microscopy approaches, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of the morphogenetic proteins SpoIVA, SpoVID, SafA and/or CotE. Significance statementBacterial spores are dormant cells that can resist to multiple stresses, including antibiotics, detergents, irradiation and high temperatures. Such resilience is an asset when spores are used for the benefit of humans, as in the case of probiotics, or a major problem for public health, food safety or biowarfare when it comes to spores of pathogenic bacteria. In this study, we combined state-of-the-art cryo-electron tomography and conventional cellular electron microscopy to provide insights into intermediate stages of spore development. Our data reveal the intracellular reorganization of the chromosome into a toroidal fibrillar structure and the complex assembly of the multi-protein, multilayered extracellular coat, shedding light on the mechanisms by which the spore acquires its incredible resistance capacities.

microbiology↗

bioRxiv2022

Electron microscopy reveals toroidal shape of master neuronal cell differentiator REST - RE1-Silencing Transcription factor

The RE1-Silencing Transcription factor (REST) is essential for neuronal differentiation. Here, we report the first 18.5-angstrom electron microscopy structure of human REST. The refined electron map suggests that REST forms a torus that can accommodate DNA double-helix in the central hole. Additionally, we quantitatively described REST binding to the canonical DNA sequence of the neuron-restrictive silencer element. We developed protocols for the expression and purification of full-length REST and the shortened variant REST-N62 produced by alternative splicing. We tested the mutual interaction of full-length REST and the splicing variant REST-N62. Revealed structure-function relationships of master neuronal repressor REST will allow finding new biological ways of prevention and treatment of neurodegenerative disorders and diseases.

biochemistry↗

bioRxiv2022

Molecular basis of indispensable accuracy of mammalian miRNA biogenesis

Mammalian Dicer is the gatekeeper into the essential gene-regulating miRNA pathway. What is committing mammalian Dicer to the miRNA pathway remains unknown. We report that Dicers highly conserved DExD/H helicase domain is the key structural element supporting accurate miRNA biogenesis. While ATPase activity of the domain is non-essential, its loss is lethal in mice. It is required during canonical miRNA biogenesis for efficient recognition, high-fidelity cleavage, and strand selection. Structure of Dicer-miRNA precursor complexes showed that the DExD/H domain acquired helicase-unrelated function defining Dicer conformations, which affect substrate loading and facilitate pre-selection of miRNA precursors. Dicer lacking the DExD/H domain favors conformations enabling reduced substrate selectivity and supporting RNA interference, a different small RNA pathway. Therefore, Dicers DExD/H domain ensures indispensable high-fidelity precursor processing of mammalian miRNAs, which constitutes a structural "mold" for adaptive miRNA evolution.

molecular biology↗

bioRxiv2020

Lipoprotein particles interact with membranes and transfer their cargo without receptors

Lipid transfer from lipoprotein particles to cells is essential for lipid homeostasis. High density lipoprotein (HDL) particles are mainly captured by cell-membrane-associated scavenger receptor class B type 1 (SR-B1) from the blood stream while low and very low density lipoprotein (LDL, VLDL) particles are mostly taken up by receptor-mediated endocytosis. However, the role of the target lipid membrane itself in the transfer process has been largely neglected so far. Here, we study how lipoprotein particles (HDL, LDL and VLDL) interact with synthetic lipid bilayers and cell-derived membranes and transfer their cargo subsequently. Employing cryo-electron microscopy, spectral imaging and fluorescence (cross) correlation spectroscopy allowed us to observe integration of all major types of lipoprotein particles into the membrane and delivery of their cargo in a receptor-independent manner. Importantly, biophysical properties of the target cell membranes change upon cargo delivery. The concept of receptor-independent interaction of lipoprotein particles with membranes helps to better understand lipoprotein particle biology and can be exploited for novel treatments of dyslipidemia diseases.

biophysics↗

bioRxiv2020

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

RNA synthesis is central to life, and RNA polymerase depends on accessory factors for recovery from stalled states and adaption to environmental changes. Here we investigated the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis RNA polymerase and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNA polymerase channels that are responsible for DNA binding and substrate delivery to the active site, thereby locking RNA polymerase in an inactive state. We show that HelD prevents non-specific interactions between RNA polymerase and DNA and dissociates transcription elongation complexes, but does not inhibit RNA polymerase binding to the initiation {sigma} factor. The liberated RNA polymerase can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that removes undesirable nucleic acids from RNA polymerase.

molecular biology↗

bioRxiv2020

Moderate prenatal ethanol exposure increases total length of L1 axons in E15.5 mice

Public health campaigns broadcast the link between heavy alcohol consumption during pregnancy and physical, cognitive, and behavioral birth defects; however, they appear less effective in deterring moderate consumption prevalent in women who are pregnant or of childbearing age. The incidence of mild Fetal Alcohol Spectrum Disorders (FASD) is likely underestimated because the affected individuals lack physical signs such as retarded growth and facial dysmorphology and cognitive/behavioral deficits are not commonly detected until late childhood. Sensory information processing is distorted in FASD, but alcohol effects on the development of axons that mediate these functions are not widely investigated. We hypothesize that alcohol exposure alters axon growth and guidance contributing to the aberrant connectivity that is a hallmark of FASD. To test this, we administered alcohol to pregnant dams from embryonic day (E) 7.5 to 14.5, during the time that axons which form the major forebrain tracts are growing. We found that moderate alcohol exposure had no effect on body weight of E15.5 embryos, but significantly increased the length of L1+ axons. Our findings support our hypothesis. Future studies will investigate cellular, molecular, and functional mechanisms that underlie these effects.

developmental biology↗

bioRxiv2019

A role for the Saccharomyces cerevisiae ABCF protein New1 during translation termination

Translation on the ribosome is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related ABCF ATPase, New1, is encoded by a non-essential gene with a cold sensitivity and ribosome assembly defect knock-out phenotype. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide ribosome assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed a combination of yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination.

biochemistry↗

bioRxiv2018

A strategy to study intrinsically mixed folded proteins: The structure in solution of ataxin-3

It has increasingly become clear over the last two decades that proteins can contain both globular domains and intrinsically unfolded regions which both can contribute to function. While equally interesting, the disordered regions are difficult to study because they usually do not crystallize unless bound to partners and are not easily amenable to cryo-electron microscopy studies. Nuclear magnetic resonance spectroscopy remains the best technique to capture the structural features of intrinsically mixed folded proteins and describe their dynamics. These studies rely on the successful assignment of the spectrum, task not easy per se given the limited spread of the resonances of the disordered residues. Here, we describe assignment of the spectrum of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We used a 42 kDa construct containing a globular N-terminal josephin domain and a C-terminal tail which comprises thirteen polyglutamine repeats within a low-complexity region. We developed a strategy which allowed us to achieve 87% assignment of the spectrum. We show that the C-terminal tail is flexible with extended helical regions and interacts only marginally with the rest of the protein. We could also, for the first time, deduce the structure of the polyglutamine repeats within the context of the full-length protein and show that it has a strong helical propensity stabilized by the preceding region.

biophysics↗