Biology subjects

Engel, B. D.

Publications and source records attributed to Engel, B. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Thylakoid plasticity primes photoprotection in diatoms

For many millions of years, diatoms have been major primary producers in marine ecosystems, supporting food webs and contributing to carbon sequestration through the export of photosynthetically fixed carbon to the deep ocean, mitigating atmospheric carbon dioxide levels and global climate. A key factor in their ecological success is their ability to rapidly adapt to fluctuating light, balancing efficient light harvesting under dim conditions with energy dissipation under excess light. However, the mechanisms behind this regulation have not been fully elucidated. This study shows that several processes contribute to dissipating excess excitation energy in diatoms exposed to light stress: (i) the detachment of the oxygen-evolving complex proteins from the PSII core, leading to energy dissipation via charge recombination, (ii) Non-Photochemical Quenching (NPQ) in the antenna, i.e., thermal dissipation of excited states and (iii) excitation energy transfer from PSII to PSI via energy spillover. The latter process is observed in parallel with high light-induced topological changes of the thylakoids that may favor transient interactions between the photosystems. Along with the accumulation of light-stress-protecting carotenoids on longer time-scales, this ensemble of photoprotective mechanisms provides a dynamic and flexible response to the light challenges expected in coastal marine habitats.

biochemistry↗

bioRxiv2026

Far-Red Light Photoacclimation Rewires Antenna-Photosystem Organization in Cyanobacterial Cells

Thylakoid membrane remodeling regulates photosynthetic activity in response to changing light, but it is unknown whether such membrane-scale control exists in cyanobacteria, which lack the spatially segregated membrane domains of green algae and plants. Using cryo-electron tomography guided by single-cell hyperspectral correlative imaging, we link spectral acclimation state to thylakoid architecture in far-red light adapting cyanobacteria. We determine the native structures of phycobilisome-photosystem II supercomplexes at subnanometer resolution and show that light-dependent molecular changes propagate across scales to restructure the photosynthetic machinery throughout the thylakoids. We further reveal persistent membrane heterogeneity after prolonged far-red light acclimation and identify a low abundance phycobilisome-photosystem I supercomplex in far-red light conditions. Together, our results frame cyanobacterial thylakoids as plastic membranes that remodel antenna-photosystem structure and organization to regulate photosynthesis.

molecular biology↗

bioRxiv2026

Lateral organization of cytochrome b6f in thylakoid membranes controls photosynthetic electron transfer efficiency

Photosynthetic electron transfer relies on the coordinated function and spatial organization of large protein complexes within the thylakoid membrane. The cytochrome b6f complex (b6f) functionally interconnects photosystem (PS) II and PSI in photosynthetic electron transfer and is equally distributed between appressed and non-appressed thylakoid membranes. Here, we investigate the functional link between the lateral distribution of b6f and efficient photosynthetic electron flow in Chlamydomonas reinhardtii. We engineered strains with stromal fusions between PetA of b6f and two fluorescent proteins (FPs) of different molecular mass: Clover and ATeam. Under oxic conditions, these strains exhibited significantly slower electron transfer rates (ETR), lower PSII quantum yields, and increased donor-side limitation of PSI. State transitions were diminished in the fusion strains, accompanied by a strong impairment of STT7-dependent function, suggesting that the presence of fused FPs at b6f interfere with STT7 function. Yet, ETR phenotypes were STT7-independent and FP fusion did not impact intrinsic b6f function. In situ cryogenic electron tomography revealed a significant depletion of b6f from appressed thylakoid membranes in the ATeam strains, while the overall membrane protein concentration remained unchanged. Overall, our data indicate that a balanced distribution of b6f between appressed and non-appressed thylakoid membranes is essential for regulating photosynthetic electron transfer, highlighting the functional importance of thylakoid molecular architecture in vivo.

plant biology↗

bioRxiv2026

Molecular organization of the Chlorella sorokiniana pyrenoid

To overcome the enzymatic limitations of Rubisco, algae operate CO2-concentrating mechanisms (CCMs) that deliver concentrated CO2 to Rubisco tightly packaged in a specialized microcompartment called a pyrenoid. Pyrenoids are globally important biomolecular condensates, but their convergent evolution means that their molecular composition and emergent architecture cannot be inferred across clades. Here we characterize the pyrenoid of the Trebouxiophyceae alga, Chlorella sorokiniana. Using cryo-electron tomography, we provide an architectural overview of the pyrenoid and visualize pyrenoid-specific protein complexes. Quantitative proteomics and Rubisco co-immunoprecipitation followed by mass spectrometry demonstrate that inorganic carbon delivery machinery is conserved across green algae but the pyrenoid structural components are not. In vitro reconstitution supports the role of two previously undescribed proteins, one in assembly of pyrenoid traversing thylakoids (putative matrix thylakoid tether; PMTT) and another in starch tethering to the Rubisco matrix (putative matrix starch tether; PMST). In Nicotiana benthamiana, PMTT localized to the thylakoid stromal lamellae and PMST to chloroplast starch granules. Our findings provide insights into the molecular logic of pyrenoid assembly; how proteins mediate condensate-membrane and condensate-starch interactions; and expands the pyrenoid plant engineering toolkit, setting the stage for engineering a Chlorella pyrenoid into plants.

plant biology↗

bioRxiv2026

Conservation and evolution of the ribosome across the photosynthetic lineage

Regulated biogenesis of photosynthetic proteins by the chloroplast ribosomes is central to plant development and environmental adaptation. Here, we describe changes to the ribosome that occurred during evolution of chloroplasts from cyanobacteria in the photosynthetic lineage based on structural, proteomic and bioinformatic analyses. We identify structural features common to oxygenic photosynthetic organisms and distinct from other bacteria, as well as the gain and loss of ribosomal proteins, rRNA modifications and hibernation factors upon endosymbiosis. We uncover structural variation among land plant lineages at sites that remodel the peptide exit tunnel and features associated with translation initiation. The data challenge the prevailing view of ribosomal features that are specific to the chloroplast by demonstrating that many originated earlier, in cyanobacteria, or later, during land plant evolution.

biochemistry↗

bioRxiv2026

Coupling of electron-bifurcation modules powers aromatic ring reduction beyond the biological redox window

Microbial degradation of ubiquitous aromatic compounds is central to the global carbon cycle and bioremediation, yet the intrinsic stability of aromatic rings poses a major barrier to their breakdown. Under strictly anaerobic conditions, class II benzoyl-CoA reductases (BCRII) catalyse the key step of this process, a Birch-like reduction of the aromatic ring to a cyclic diene at a tungsten cofactor. This reaction operates beyond the redox limits of conventional biological electron transfer, yet the mechanism by which BCRII generates such extreme reducing power has remained unclear. Here, high-resolution cryo-electron microscopy, in situ cryo-electron tomography, and enzymatic analyses reveal that the one-MDa BCRII complex from Geobacter metallireducens links two distinct flavin-based electron-bifurcation modules, previously characterised in hydrogenases and heterodisulfide reductases, to drive aromatic ring reduction. Reduced ferredoxin and NADH deliver electrons through sequential confurcation and bifurcation to the catalytic site, while cryo-electron tomography of native cells identifies electron-transferring flavoprotein as the second acceptor directing high-potential electrons into the respiratory chain. These results show that modular FBEB units can be hierarchically assembled to extend metabolic redox capacity, highlighting their versatility as adaptable components for electron transfer pathways.

biochemistry↗

bioRxiv2026

Molecular architecture of the ciliary base in mammalian multiciliated cells

Multiciliated epithelial cells (MCCs) generate tens to hundreds of motile cilia to drive fluid flow in diverse physiological contexts. While the axonemal structure of motile cilia has been described extensively in recent years, the molecular architecture of the transition zone, basal body, and surrounding ciliary environment of MCCs remain more elusive. Here, we use cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) to obtain in situ 3D views of the ciliary base within intact MCCs from mammalian trachea, complemented by in situ cross-linking mass spectrometry (XL/MS) and ultrastructure expansion microscopy (U-ExM) for molecular identification. Our data reveal spatially-defined modifications of microtubule architecture from the proximal centriole to the early axoneme, including transition zone-specific features such as an A-B linker bridging microtubule doublets and a helical assembly of microtubule inner proteins (MIPs). We show that the ciliary necklace, a feature observed in many motile cilia, is spatially aligned with the transition zone and quantify its regular organization within the membrane. Our in situ data capture rarely observed events, including intraflagellar transport (IFT) trains connecting to ciliary vesicles tethered to undocked centrioles. The surrounding ciliary environment contains intermediate filaments that encircle the basal bodies and bundled actin filaments that elaborate microvilli structures between the cilia. Integration of XL/MS and U-ExM identified novel microtubule associated proteins (MAPs), MIPs, and membrane-associated proteins localized to these distinct subdomains. This work provides a molecular and structural map of the mammalian MCC ciliary base, revealing architectural principles that underlie its assembly, organization, and function.

cell biology↗

bioRxiv2026

Stoic: Fast and accurate protein stoichiometry prediction

MotivationProtein complexes are central to cellular function, but experimental determination of their structures remains challenging. Structure prediction methods require prior knowledge of stoichiometry - the number of copies of each protein entity within a complex. Current approaches rely on computationally expensive brute-force methods that run structure prediction on multiple stoichiometry combinations, often with limited accuracy. ResultsWe introduce Stoic, a method that uses protein language model embeddings to predict protein complex stoichiometry. Our approach learns to identify interface residues that participate in protein-protein interactions, rather than relying on global sequence features. By integrating these interface-aware embeddings into a graph neural network, Stoic achieves fast and accurate stoichiometry prediction for both homomeric and heteromeric targets. AvailabilitySource code for inference and training along with web versions are available in the repository at https://github.com/PickyBinders/stoic. Contactjanani.durairaj@unibas.ch

bioinformatics↗

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↗

bioRxiv2026

Chloroplast-encoded small subunit extensions reshape the Chlamydomonas chlororibosome

Chloroplast ribosomes (chlororibosomes) synthesize the core protein components of the photosynthetic apparatus, yet their structural diversity outside flowering plants remains largely unexplored. Here, we combine in situ cryo-electron tomography (cryo-ET) with single-particle cryo-electron microscopy (cryo-EM) to determine the structure of the chlororibosome from the unicellular green alga Chlamydomonas reinhardtii. Subtomogram averaging of chlororibosomes in their native environment, resolved to [~]5 [A] resolution and in distinct translational states, reveals particles both free in the stroma and loosely tethered to thylakoid membranes. These in situ reconstructions uncover an additional "arm" domain on the small subunit. High-resolution single-particle reconstruction of isolated chlororibosomes to [~]2.5 [A], in states bound either to the inhibitory translation factor pY or to a nascent chain-linked P-site tRNA, reveals that this domain is built primarily from extensive chloroplast-encoded insertions and extensions of conserved small subunit proteins, supported by chlororibosome-specific ribosomal proteins. The arm domain is located around the mRNA entry and exit channels, suggesting a role in stabilizing the mRNA trajectory through the small subunit and organizing chloroplast polysomes. Together, these data reveal unexpected structural variation of algal chlororibosomes and suggest that chloroplast translation has diversified substantially even among relatively closely related photosynthetic lineages.

molecular biology↗

bioRxiv2025

Icecream: High-Fidelity Equivariant Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) visualizes 3D cellular architecture in near-native states. Recent deep-learning methods (CryoCARE, IsoNet, DeepDeWedge, CryoLithe) improve denoising and artifact correction, but performance remains limited by very low signal-to-noise ratio, a restricted angular range ( missing wedge), and the lack of ground truth. Here, we present Icecream, which follows the broad template of earlier self-supervised approaches, but treats symmetry in a way consistent with the recent equivariant imaging framework (Chen et al., 2021). Coupled with several engineering refinements, including mixed-precision arithmetic, Icecream achieves substantially better denoising and more reliable missing-wedge recovery, while reducing training and inference time relative to comparable baselines. Across diverse experimental datasets, we observe consistent gains in reconstruction quality, both visually and as quantified by Fourier shell correlation (FSC). Our framework extends to any tomography problem that provides two statistically independent reconstructions of the same volume; in cryo-ET these are obtained by dose splitting or angular partitioning of the tilt series.

bioinformatics↗

bioRxiv2025

The Luminal Ring Protein C2CD3 Acts as a Radial In-to-Out Organizer of the Distal Centriole and Appendages

Centrioles are polarized microtubule-based structures with appendages at their distal end that are essential for cilia formation and function. The protein C2CD3 is critical for distal appendage assembly, with mutations linked to orofaciodigital syndrome and other ciliopathies. However, its precise molecular role in appendage recruitment remains unclear. Using Ultrastructure Expansion Microscopy (U-ExM), iterative U-ExM, and in situ cryo-electron tomography (cryo-ET), we reveal that C2CD3 adopts a radially symmetric 9-fold organization within the centrioles distal lumen. We show that the C-terminal region of C2CD3 localizes close to a [~]100 nm luminal ring structure consisting of [~]27 nodes, while its N-terminal region localizes close to a hook-like structure that attaches to the A-microtubule as it extends from the centriole interior to exterior. This hook structure is adjacent to the DISCO complex (MNR/CEP90/OFD1), which marks future appendage sites. C2CD3 depletion disrupts not only the recruitment of the DISCO complex via direct interaction with MNR but also destabilizes the luminal ring network composed of C2CD3/SFI1/centrin-2/CEP135/NA14, as well as the distal microtubule tip protein CEP162. This reveals an intricate "in-to-out" molecular hub connecting the centriolar lumen, distal microtubule cap, and appendages. Although C2CD3 loss results in shorter centrioles and appendage defects, key structural elements remain intact, permitting continued centriole duplication. We propose that C2CD3 forms the luminal ring structure and extends radially to the space between triplet microtubules, functioning as an architectural hub that scaffolds the distal end of the centriole, orchestrating its assembly and directing appendage formation.

cell biology↗

bioRxiv2024

Towards community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii

In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate structures of biomolecules in their native context. However, challenges remain in the efficient production of large-scale cryo-ET datasets, as well as the community sharing of this information-rich data. Here, we applied a cryogenic plasma-based focused ion beam (cryo-PFIB) instrument for high-throughput milling of the green alga Chlamydomonas reinhardtii, a useful model organism for in situ visualization of numerous fundamental cellular processes. Combining cryo-PFIB sample preparation with recent advances in cryo-ET data acquisition and processing, we generated a dataset of 1829 reconstructed and annotated tomograms, which we provide as a community resource to drive method development and inspire biological discovery. To assay the quality of this dataset, we performed subtomogram averaging (STA) of both soluble and membrane-bound complexes ranging in size from >3 MDa to [~]200 kDa, including 80S ribosomes, Rubisco, nucleosomes, microtubules, clathrin, photosystem II, and mitochondrial ATP synthase. The majority of these density maps reached sub-nanometer resolution, demonstrating the potential of this C. reinhardtii dataset, as well as the promise of modern cryo-ET workflows and open data sharing towards visual proteomics.

cell biology↗

bioRxiv2024

Molecular architecture of thylakoid membranes within intact spinach chloroplasts

Thylakoid membranes coordinate the light reactions of photosynthesis across multiple scales, coupling the architecture of an elaborate membrane network to the spatial organization of individual protein complexes embedded within this network. Previously, we used in situ cryo-electron tomography (cryo-ET) to reveal the native thylakoid architecture of the green alga Chlamydomonas reinhardtii [1] and then map the molecular organization of these thylakoids with single-molecule precision [2]. However, it remains to be shown how generalizable this green algal blueprint is to the thylakoids of vascular plants, which possess distinct membrane architecture subdivided into grana stacks interconnected by non-stacked stromal lamellae. Here, we continue our cryo-ET investigation to reveal the molecular architecture of thylakoids within intact chloroplasts isolated from spinach (Spinacia oleracea). We visualize the fine ultrastructural details of grana membranes, as well as interactions between thylakoids and plastoglobules. We apply and further develop AI-based computational approaches for automated membrane segmentation and membrane protein picking [3], enabling us to quantify the organization of photosynthetic complexes within the plane of the thylakoid membrane and across adjacent stacked membranes. Our analysis reveals that, despite different 3D architecture, the molecular organization of thylakoid membranes in vascular plants and green algae is strikingly similar. In contrast to isolated plant thylakoids, where semi-crystalline arrays of photosystem II (PSII) appear to hold some membranes together, we find in intact chloroplasts that PSII is non-crystalline and has uniform concentration both within the membrane plane and across stacked grana membranes. Similar to C. reinhardtii, we observe strict lateral heterogeneity of PSII and PSI at the boundary between appressed and non-appressed thylakoid domains, with no evidence for a distinct grana margin region where these complexes have been proposed to intermix. Based on these measurements, we support a simple two-domain model for the molecular organization of thylakoid membranes in both green algae and plants.

plant biology↗

bioRxiv2024

In-cell architecture of the mitochondrial respiratory chain

Mitochondria produce energy through oxidative phosphorylation, carried out by five membrane-bound complexes collectively known as the respiratory chain. These complexes work in concert to transfer electrons and pump protons, leading to ATP regeneration. The precise organization of these complexes in native cells is debated, notably their assembly into higher-order supercomplexes called respirasomes. Here, we use in situ cryo-electron tomography to visualize the native structures and organization of several major mitochondrial complexes inside Chlamydomonas reinhardtii cells. ATP synthases and respiratory complexes are segregated into curved and flat crista membrane domains, respectively. Respiratory complexes I, III, and IV assemble into a single type of respirasome, from which we determined a native 5 [A]-resolution structure showing the binding of electron carrier cytochrome c. Combined with single-particle cryo-electron microscopy reconstruction at 2.4 [A] resolution, we assemble a detailed model of how the respiratory complexes interact with each other inside native mitochondria.

molecular biology↗

bioRxiv2024

Surforama: interactive exploration of volumetric data by leveraging 3D surfaces

MotivationVisualization and annotation of segmented surfaces is of paramount importance for studying membrane proteins in their native cellular environment by cryogenic electron tomography (cryo-ET). Yet, analyzing membrane proteins and their organization is challenging due to their small sizes and the need to consider local context constrained to the membrane surface. ResultsTo interactively visualize, annotate, and analyze proteins in cellular context from cryo-ET data, we have developed Surforama, a Python package and napari plugin. For interactive visualization of membrane proteins in tomograms, Surforama renders the local densities projected on the surface of the segmentations. Suforama additionally provides tools to annotate and analyze particles on the membrane surfaces. Finally, for compatibility with other tools in the cryo-ET analysis ecosystem, results can be exported as RELION-formatted STAR files. As a demonstration, we performed subtomogram averaging and neighborhood analysis of photosystem II proteins in thylakoid membranes from the green alga Chlamydomonas reinhardtii. Availability and implementationPython package, code and examples are available at: https://github.com/cellcanvas/surforama

bioinformatics↗

bioRxiv2024

MemBrain v2: an end-to-end tool for the analysis of membranes in cryo-electron tomography

Cryo-electron tomography (cryo-ET) provides unique insights into macromolecular complexes in their native environments, yet membrane analysis remains a major bottleneck due to low signal-to-noise ratios, missing wedge artifacts, and the complexity of membrane-associated proteins. Existing tools often require extensive manual annotation, struggle with generalization across datasets, and lack integrated solutions for segmentation, protein localization, and quantitative analysis. We introduce MemBrain v2, a deep learning-enabled framework that unifies these tasks into a streamlined pipeline. MemBrain-seg leverages a diverse, collaboratively generated training dataset and specialized model training strategies to achieve generalizable membrane segmentation across variable tomographic conditions. MemBrain-pick enables data-efficient localization of membrane-bound proteins by integrating geometric constraints with deep learning, reducing the need for extensive manual annotation. MemBrain-stats provides quantitative insights into protein distributions, computing spatial metrics to analyze intra-membrane particle organization. MemBrain v2 integrates seamlessly into cryo-ET workflows, providing an accessible and structured approach to membrane analysis. The full package is available at https://github.com/CellArchLab/MemBrain-v2.

bioinformatics↗

bioRxiv2023

Diatom pyrenoids are encased in a protein shell that enables efficient CO2 fixation

Pyrenoids are subcompartments of algal chloroplasts that concentrate Rubisco enzymes and their CO2 substrate, thereby increasing the efficiency of carbon fixation. Diatoms perform up to 20% of global CO2 fixation, but their pyrenoids remain poorly characterized at a molecular level. Here, we used in vivo photo-crosslinking to catalogue components of diatom pyrenoids and identified a pyrenoid shell (PyShell) protein, which we localized to the pyrenoid periphery of both the pennate diatom, Pheaodactylum tricornutum, and the centric diatom, Thalassiosira pseudonana. In situ cryo-electron tomography (cryo-ET) revealed that the pyrenoids of both diatom species are encased in a lattice-like protein sheath. Disruption of PyShell expression in T. pseudonana resulted in the absence of this protein sheath, altered pyrenoid morphology, and a high-CO2 requiring phenotype, with impaired growth and reduced carbon fixation efficiency under standard atmospheric conditions. Pyrenoids in mutant cells were fragmented and lacked the thylakoid membranes that normally traverse the Rubisco matrix, demonstrating how the PyShell plays a guiding role in establishing pyrenoid architecture. Recombinant PyShell proteins self-assembled into helical tubes, enabling us to determine a 3.0 [A]-resolution PyShell structure. We then fit this in vitro structure into an in situ subtomogram average of the pyrenoids protein sheath, yielding a putative atomic model of the PyShell within diatom cells. The structure and function of the diatom PyShell provides a new molecular view of how CO2 is assimilated in the ocean, a crucial biome that is on the front lines of climate change.

cell biology↗