Biology subjects

Mattei, S.

Publications and source records attributed to Mattei, S..

12 recordsLinked to original sources

bioRxiv2026

Super-Resolution Processing Enables In Situ Subtomogram Averaging Beyond the Nyquist Limit

In situ subtomogram averaging (STA) for high-resolution structure determination requires fine pixel sampling, forcing a trade-off between achievable resolution and the field of view (FOV) recorded in each tomogram. Electron-event representation (EER) data can be rendered at half the physical pixel size, but whether super-resolution processing can recover useful information beyond the physical Nyquist limit from noisy in situ data remains unclear. Here, we show that super-resolution processing of EER data recovers biologically interpretable information beyond a physical Nyquist limit of 4.78 A in two in situ datasets. STA of unidentified virus particles in a Drosophila melanogaster larva yielded a 3.42 A inner capsid reconstruction, enabling de novo model building and identification of the particles as Drosophila A virus. In situ STA of ribosomes in Saccharomyces cerevisiae reached 3.18 A, with local resolution extending to 2.78 A. EER acquisition therefore provides greater flexibility in choosing the acquisition magnification: existing datasets can be re-rendered at the super-resolution pixel size when physical sampling becomes limiting, while lower-magnification acquisition can be considered when a larger FOV improves target capture or cellular context.

molecular biology↗

bioRxiv2026

PinCorr: A high-pressure freezing carrier with intrinsic landmarks for cryo-correlative light and electron microscopy

Cryo-correlative light and electron microscopy methods enable targeted structural analysis of fluorescently labelled features in vitrified specimens. However, correlative workflows on high-pressure frozen samples often remain challenging due to the lack of persistent landmarks for reliable sample tracking and image registration between different microscopes. Standard high-pressure freezing carriers provide little intrinsic reference information, as the exposed sample surface is often smooth and rotationally ambiguous, complicating localisation of regions of interest across imaging platforms. Here, we introduce PinCorr, a 3-mm high-pressure freezing carrier with an integrated coordinate system formed by four asymmetrically arranged pillars with distinct geometries. These built-in landmarks remain visible after freezing and provide a stable, sample-independent reference frame for orientation and correlation between cryo-fluorescence microscopy and electron microscopy. We show that PinCorr supports fluorescence-guided cryo-volume imaging, serial lift-out for cryo-electron tomography and freeze-substitution workflows followed by room-temperature on-section correlation. PinCorr thus provides a hardware-based approach to establishing a persistent spatial reference frame in HPF-based correlative imaging workflows for thick and multicellular specimens.

Molecular Biology↗

bioRxiv2026

LowDoseWizard - rapid and standardised setup of low-dose cryo-TEM imaging in SerialEM

Structure elucidation of biological macromolecules by single particle cryogenic electron microscopy (SPA cryo-EM) or cryogenic electron tomography (cryo-ET) relies on low-dose imaging on cryogenic transmission electron microscopes (cryo-TEMs). Routine microscope setup remains technically demanding and can be time-consuming, particularly for inexperienced or infrequent users. We present LowDoseWizard, a guided workflow implemented in SerialEM that enables rapid and standardised setup of cryo-TEM imaging conditions. From minimal user input, the workflow configures microscope optics, camera parameters and image shift settings for all low-dose imaging states, and guides the user through key daily alignment procedures including beam shift offset calibration, objective lens astigmatism correction and coma-free alignment. The workflow is organised into modular routines that can be executed sequentially or independently, while microscope-specific acquisition parameters are defined in editable configuration files, allowing flexible adaptation to different instruments without modification of the core scripts. Across user sessions on three microscopes at EMBL Heidelberg, the complete setup required on average less than 15 minutes. To assess whether predefined imaging conditions generated by the workflow are compatible with high-resolution data collection, we acquired apoferritin data on a 200 kV Glacios and a 300 kV Titan Krios. These datasets yielded reconstructions at 1.62 [A] and 1.09 [A] resolution, respectively, demonstrating that rapid, guided setup can support near-atomic and atomic-resolution single particle cryo-EM. LowDoseWizard lowers the barrier to robust cryo-TEM setup, reduces the time spent on routine parameter selection and alignment, and helps users focus on sample-specific aspects of data acquisition such as target selection. The workflow should be particularly valuable in shared instrumentation environments, where accessibility, reproducibility and efficient microscope use are critical.

molecular biology↗

bioRxiv2026

A Novel Eukaryotic Ribosome Factor Enables Translation Restart Following Cellular Dormancy

Dormancy is a survival strategy employed by all domains of life to withstand prolonged nutrient deprivation and environmental stress that is marked by a global shutdown of protein synthesis. However, the molecular mechanisms driving ribosome inactivation and reactivation during and after dormancy in eukaryotes remain poorly understood. Here, we identify SNOR, a novel SBDS-like ribosome-associated factor in Schizosaccharomyces pombe, that is upregulated and associates with ribosomes during induced dormancy triggered by glucose depletion. SNOR contributes to protein synthesis repression by binding the ribosome to probe the peptidyl transferase center (PTC), block tRNA-binding sites, and cap the polypeptide exit tunnel (PET). Importantly, we show that SNOR is essential for the restart of protein synthesis upon glucose reintroduction and exit from dormancy. SNOR is evolutionarily conserved and specifically upregulated in response to glucose stress in fungi. These findings reveal a previously unrecognized ribosome-associated factor that links glucose stress and cellular dormancy to surveillance of protein synthesis and highlight the power of in situ structural biology to uncover stress-responsive regulators of translation.

biophysics↗

bioRxiv2026

An Advanced Mobile Laboratory to enable field-based microbial ecology and cell biology across scales

Microbial biodiversity is central to ecosystem function, yet mechanistic insights into the cell biology of environmental organisms remain limited. The underlying challenges are twofold: most microbes remain uncultivable, and a persistent gap exists between field sampling and laboratory analyses. Here, we introduce the Advanced Mobile Laboratory (AML), a field-deployable platform that integrates confocal microscopy, image-enabled cell sorting, and cryo-preparation for expansion and electron microscopy. This setup enables immediate, standardized processing and analysis of environmental communities directly at the sampling site. We demonstrate its capability using marine eukaryotic plankton, showing how the AML enables multiscale investigations, from live imaging of natural communities to enabling ultrastructural and single-cell omics analyses, while minimizing sample degradation and enabling on-site experimentation. By bringing high-end sample preparation and analytical capacity into the field, the AML enables studying life in its natural context to mechanistically understand lifes diversity in the environment.

cell biology↗

bioRxiv2025

The In Situ Structure and Distribution of V-ATPase in C. elegans Apical Membrane Stacks

The cuticle of Caenorhabditis elegans is an apical extracellular matrix composed primarily of collagens which protects the organism from external stresses and facilitates mobility. The cuticle directly contacts several epithelial cell structures including regular, repeating folds of the apical plasma membrane, known as apical membrane stacks (AMSs). Here, we examine the structure of AMSs using in situ cryo-electron tomography (cryo-ET). We find that V-ATPases are distributed across the cytoplasmic-facing membranes of the AMSs. We then determine the in situ structure of V-ATPase and show that it is highly conserved relative to other eukaryotic V-ATPases. Analysis of the distribution of V-ATPases on the AMSs shows that they form clusters in which individual V-ATPases are tightly packed but do not oligomerise. Our data also show that complete, fully assembled V-ATPases line the AMSs. Previous studies have established that V-ATPases can only act as ATP-dependent proton pumps in their fully assembled state. Our findings indicate that V-ATPases arranged along the AMSs pump protons directly into the extracellular matrix, suggesting that a primary function of AMSs is to acidify the cuticle.

molecular biology↗

bioRxiv2025

Dual apical methyltransferases orchestrate motility initiation in apicomplexan parasites

Apicomplexan parasites such as Toxoplasma gondii initiate motility through rapid, spatially confined cytoskeletal activation at their apical end. While calcium-, lipid-, and kinase-based signalling pathways have been partially elucidated, how these cues are translated into mechanical force remains unclear. Here, we uncover a dual methyltransferase mechanism that orchestrates this process. We identify PCKMT, a preconoidal ring-associated lysine methyltransferase, as an essential upstream regulator of motility. PCKMT anchors the actin nucleator Formin-1 (FRM1) at the conoid, enabling conoid protrusion and F-actin assembly. Loss of PCKMT abolishes FRM1 recruitment, blocks conoid extrusion, and arrests invasion and egress despite preserved conoid structure. In contrast, the apical methyltransferase AKMT, previously linked to motility through recruitment of the glideosome-associated connector (GAC), acts downstream, disengaging from the conoid upon activation and likely promoting GAC-dependent force transmission. PCKMT depletion prevents AKMT translocation, revealing that actin assembly and lysine methylation are mechanistically coupled. Together, these findings define a two-step methylation checkpoint that coordinates actin nucleation with force propagation, uncovering methylation as a central regulatory axis for motility initiation in apicomplexan parasites.

microbiology↗

bioRxiv2024

EasyGrid: A versatile platform for automated cryo-EM sample preparation and quality control

Imaging biological macromolecules in their native state with single-particle cryo-electron microscopy (cryo-EM) or in situ cryo-electron tomography (cryo-ET) requires optimized approaches for the preparation and vitrification of biological samples. Here, we describe EasyGrid, a versatile technology enabling systematic, tailored and advanced sample preparation for cellular and structural biology. This automated, standalone platform combines in-line plasma treatment, microfluidic dispensing, blot-less sample spreading, jet-based vitrification and on-the-fly grid quality control using light interferometry to streamline cryo-EM sample optimization. With EasyGrid, we optimized grid preparation for different purified macromolecular complexes and subsequently determined their structure with cryo-EM. We also demonstrated how the platform allows better vitrification of large, mammalian cells compared to standard plunge-freezing. Automated sample preparation with EasyGrid establishes an advanced, high-throughput platform for both single-particle cryo-EM and cellular cryo-ET sample preparation.

molecular biology↗

bioRxiv2023

Hibernating ribosomes tether to mitochondria as an adaptive response to cellular stress during glucose depletion

Cell survival under nutrient-deprived conditions relies on cells ability to adapt their organelles and to rewire their metabolic pathways. In the fission yeast Schizosaccharomyces pombe, nutrient depletion is an unfavorable condition for protein synthesis and triggers a response characterized by mitochondrial fragmentation and the sequestration of cytosolic ribosomes on mitochondria. The molecular mechanism underlying ribosomal sequestration remains elusive. In this study, we performed time-lapse in situ cryo-electron tomography and cryo-electron microscopy complemented by biochemical experiments to elucidate the molecular details of this adaptive response. Our analysis indicate that upon glucose depletion protein synthesis is halted, causing ribosomes to enter an inactive state characterized by a conformational change that obstructs the peptidyl transferase center. Our in situ experiments reveal the presence of oligomeric arrays of hibernating ribosomes tethered to the mitochondrial surface. Surprisingly, ribosomes bind to the outer mitochondrial membrane via the small ribosomal subunit, an interaction facilitated by the ribosomal protein RACK1-orthologue Cpc2. Our experiments show that ribosome tethering is important for cell survival under glucose depletion conditions. This study broadens our understanding of the cellular adaptations triggered by nutrient scarcity and the underlying molecular mechanisms that regulate cell quiescence.

molecular biology↗

bioRxiv2022

The translating bacterial ribosome at 1.55 A resolution by open access cryo-EM

Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome1-4. This complex macromolecular machine is the target of important antimicrobial drugs5, an integral line of defence against infectious diseases. Here, we describe how open access to state-of-the-art cryogenic electron microscopy facilities combined with bespoke user support offered by the newly established EMBL Imaging Centre enabled structural determination of the translating ribosome from Escherichia coli at 1.55 [A] resolution. The obtained structures allow for direct determination of the rRNA sequence to identify ribosome polymorphism sites in the E. coli strain used in this study and enables interpretation of the ribosomal active and peripheral sites at unprecedented resolution. This includes scarcely populated chimeric hybrid states of the ribosome engaged in several tRNA translocation steps resolved at ~2 [A] resolution. The current map not only improves our understanding of protein synthesis but also allows for more precise structure-based drug design of antibiotics to tackle rising bacterial resistance.

molecular biology↗

bioRxiv2022

Mechanism of RNA Polymerase I selection by transcription factor UAF

Pre-ribosomal RNA is selectively transcribed by RNA Polymerase (Pol) I in eukaryotes. The yeast transcription factor Upstream Activating Factor (UAF) represses Pol II transcription and mediates Pol I preinitiation complex (PIC) formation during the early stages of transcription initiation at the 35S ribosomal RNA gene. To unravel the DNA recognition and Pol I selection mechanisms of UAF, we determined the structure of UAF bound to native promoter DNA and transcription factor TBP. We found that UAF recognizes DNA using a hexameric histone-like scaffold with markedly different interactions than the nucleosome and the histone-fold-rich TFIID. UAF strategically sequesters TBP from DNA and Pol II/III-specific factors, and positions it for Core Factor binding, supporting Pol I recruitment. Our findings therefore reveal the molecular basis of Pol I selection for ribosome biogenesis. As well, they reveal an unexpected potential within the histone fold as a motif for specific protein-DNA interactions inside the cell.

molecular biology↗

bioRxiv2020

Cone-shaped HIV-1 capsids are transported through intact nuclear pores

Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrate that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, coneshaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

microbiology↗