Biology subjects

Jensen, R. K.

Publications and source records attributed to Jensen, R. K..

10 recordsLinked to original sources

bioRxiv2025

Structural reorganization underlying stress-induced cytoplasmicsolidification in yeast

Cells employ diverse strategies to rapidly adapt to sudden environmental changes. In yeast, cytoprotective solidification in response to starvation and energy depletion (ED) has been reported and associated with extensive mesoscale macromolecular assembly. Yet, the structural and molecular basis underlying such whole-cell level liquid-to-solid phase transitions remain unknown. Here, we use cryo-electron tomography to characterize the subcellular organization of intact yeast cells exposed to ED and other stressors, and to untangle the effects of molecular crowding versus cytoplasmic acidification previously suggested to underpin solidification. We visualize self-assembly of macromolecules and complexes into ordered assemblies and condensates under ED, and quantify ribosome and polysomes concentrations to probe changes in cytoplasmic crowding. Combined with live-cell microscopy, we pinpoint supramolecular assembly induced by acidification, rather than a uniform increase in intracellular crowding, as the structural basis of cytoplasmic solidification that supports yeast cells adaptation in response to environmental stresses.

molecular biology↗

bioRxiv2025

CryoSiam: self-supervised representation learning for automated analysis of cryo-electron tomograms

Cryo-electron tomography (cryo-ET) enables visualization of macromolecular complexes in their native cellular context, but interpretation remains challenging due to high noise levels, missing information, and lack of ground-truth data. Here, we present CryoSiam (CRYO-electron tomography SIAMese networks), an open-source framework for self-supervised representation learning in cryo-ET. CryoSiam learns hierarchical representations of tomographic data spanning both voxel-level and subtomogram-level information. To train CryoSiam, we generated CryoETSim (CRYO-Electron Tomography SIMulated), a synthetic dataset that systematically models defocus variation, sample thickness, and molecular crowding. CryoSiam trained models transfer directly to experimental data without fine-tuning and support key aspects of cryo-ET data analysis, including tomogram denoising, segmentation of subcellular structures, and macromolecular detection and identification across both prokaryotic and eukaryotic systems. Publicly available pretrained models and the CryoETSim dataset provide a foundation for scalable and automated cryo-ET analysis.

bioinformatics↗

bioRxiv2025

Single-cell visual proteomics of a minimal bacterium reveals structural coordination in gene expression

Translation is a central process in gene expression. Regulation of this process is complex, depends on factors that include cell state and the subcellular environment, and is subject to modulation via crosstalk to processes like transcription or translocation. Here, we used cryo-electron tomography of native and antibiotic-perturbed Mycoplasma pneumoniae cells to resolve 140 maps that recapitulate bacterial translation during the initiation, elongation, termination, and recycling phases. We visualized multiple transcription-translation complexes, allowing us to propose a threading-based translation reinitiation mechanism, and to provide structural evidence for a long-hypothesized supercomplex that coordinates transcription, translation, and membrane attachment. We further resolved abundant membrane-associated large ribosomal subunits, and suggest that dissociation from membranes depends on the conditional initiation of new translation, consistent with a conserved mechanism in mammalian cells. This work visualizes the multilayered control of bacterial translation and demonstrates the power of in-cell structural biology to investigate regulatory circuits in gene expression.

molecular biology↗

bioRxiv2025

Helfrich Monte Carlo Flexible Fitting: physics-based, data-driven cell-scale simulations

Simulating entire cells represents the next frontier of computational biology. Achieving this goal requires methods that accurately describe cellular membranes across spatial and temporal scales. Although three-dimensional electron microscopy enables detailed membrane visualization, limitations on acquisition geometry, data quality, and field-of-view often result in fragmented membrane representations incompatible with simulations. To resolve this, here we introduce Helfrich Monte Carlo Flexible Fitting (HMFF), an approach that integrates experimental density data into physical simulations to determine membrane structure. Through the accompanying Mosaic software platform, we apply HMFF to influenza virus particles, Mycoplasma pneumoniae cells, and entire eukaryotic organelles. The resulting models enable multi-scale simulations spanning millions of lipids and proteins at experimentally determined positions, support quantitative morphological analysis, assess uncertainty in membrane localization, and reveal physical effects implicit in the data. Together, these capabilities establish a foundation for data-driven whole-cell simulations.

biophysics↗

bioRxiv2025

In-cell discovery and characterization of a non-canonical bacterial protein translocation-folding complex

Cryo-electron tomography has emerged as powerful technology for in-cell structural biology, and in combination with breakthroughs in protein structure prediction, offers a unique opportunity for illuminating functions of previously uncharacterized macromolecular complexes. Here, we used the genome-reduced bacterium Mycoplasma pneumoniae as a minimal cell model, and determined in-cell maps of an unknown complex located at the cell surface. By combining proteomics, structure prediction, and bioinformatics, we identified the complex to include the conserved Sec-translocon and an extracellular dome-like structure largely formed by three uncharacterized proteins (Mdps). The Mdps show structural homology to a periplasmic ATP-independent foldase, PrsA, with Mdp444 retaining key catalytic residues. Our study provides the first sub-nanometer resolution maps of the complete bacterial Sec-translocation machinery, depicting several conformational states that allow us to suggest a model for translocation initiation, and revealing its coordination with a novel, extracellular, protein-folding system.

molecular biology↗

bioRxiv2024

Cryo-EM analysis of complement C3 reveals a reversible major opening of the macroglobulin ring

The C3 protein is the central molecule within the complement system and undergoes pattern-recognition-dependent proteolytic activation to C3b in the presence of pathogens and damage-associated patterns. Spontaneous pattern-independent activation of C3 occurs via hydrolysis, resulting in C3(H2O). However, the structural details of C3 hydrolysis remain elusive. Here, we show that the conformation of the C3(H2O) analog, C3MA, in which the C3 thioester is broken by aminolysis is indistinguishable from C3b except for the 77-residue anaphylatoxin (ANA) domain. In contrast, the reaction intermediate C3* formed during C3 adopts a dynamic conformation dramatically different from both C3 and C3MA/C3b. In C3*, unlocking of the macroglobulin (MG) 3 domain creates a large opening in the MG-ring through which the ANA domain translocates. In support of this mechanism, C3MA formation is inhibited by an MG3/MG4-interface-specific nanobody and prevented by linking the ANA domain to the C3 {beta}-chain. Our study reveals an unexpected dynamic behavior of C3 where an exceptional conformational change allows the translocation of an entire domain through a large dynamic opening. These results form the basis for elucidation of the in vivo contribution of C3 hydrolysis to complement activation and offer a rational approach for modulation of C3(H2O) with the potential for preventing complement activation caused by intravascular hemolysis and surface contacts.

biochemistry↗

bioRxiv2023

ColabSeg: An interactive tool for editing, processing, and visualizing membrane segmentations from cryo-ET data

Cellular cryo-electron tomography (cryo-ET) has emerged as a key method to unravel the spatial and structural complexity of cells in their near-native state at unprecedented molecular resolution. To enable quantitative analysis of the complex shapes and morphologies of lipid membranes, the noisy three-dimensional (3D) volumes must be segmented. Despite recent advances, this task often requires considerable user intervention to curate the resulting seg-mentations. Here, we present ColabSeg, a Python-based tool for processing, visualizing, cleaning, and fitting membrane segmentations from cryo-ET data for downstream analysis. ColabSeg makes many well-established algorithms for point-cloud processing easily available to the broad community of structural biologists for applications in cryo-ET through its graphical user interface (GUI). We demonstrate the usefulness of the tool with a range of use cases and biological examples. Finally, for a large Mycoplasma pneumoniae dataset of 50 tomograms, we show how ColabSeg enables high-throughput membrane segmentation, which can be used as valuable training dataset for fully automated convolutional neural network (CNN)-based segmentation.

bioinformatics↗

bioRxiv2023

The dynamics of hinge flexibility in receptor bound immunoglobulin E revealed by electron microscopy

Immunoglobulin E is a mammal specific antibody isotype supporting the immune response against parasites and venoms, but also a driver of allergic responses. Prior studies have defined the conformation of the IgE Fc fragment bound to the cell surface receptor Fc{varepsilon}RI and the dynamic properties of the IgE Fc. It remains unknown, how these prior studies translate to the complex of a full antibody including the Fab arms with the receptor. Here we show that in a cryo-EM structure of the IgE Fc{varepsilon}RI complex, IgE adopts a T-like conformation where the antigen binding Fab arms may be parallel to the cell membrane. Two additional conformations are captured in negative stain EM (ns-EM) where the arrangements of the Fab arms differ from the cryo-EM conformation. Small angle scattering data favors the Fc{varepsilon}RI bound IgE conformation observed by cryo-EM, but the major IgE conformation observed by ns-EM possibly may also occur. In all observed conformations of Fc{varepsilon}RI bound IgE, one Fab arm is fixed relative to the IgE Fc moiety whereas the second Fab may alternate its position. Introduction of flexibility in the Fab-Fc hinge diminishes the biological activity of IgE demonstrating a functional role for the observed defined Fab-Fc hinge conformations. Our data show the organization of a full size antibody on its receptor and reveal a new layer of dynamics in Fc{varepsilon}RI bound IgE on top of the well established spectrum of IgE Fc conformations. Development of novel anti-IgE therapeutics may take into account these distinct Fc{varepsilon}RI bound IgE conformations. Significance statementIgE represents a canonical antibody isotype and is a key molecule for the allergic immune response to environmental triggers driven by mast cells and basophils. The requirements for efficient mediation of IgEs effects are not fully understood. Here we elucidate the structure of the entire IgE in complex with its high affinity receptor and identify two clearly distinct and dominant conformations, in which one of the Fab arms is fixed relative to the Fc domains. Enforcing IgE flexibility impacts the biological function with potential consequences for the allergic response. This unique behavior makes IgE different from all other isotypes and its understanding sheds light on the allergenic activation of the immune response.

immunology↗

bioRxiv2021

Structure of the integrin receptor M2 headpiece in complex with a function-modulating nanobody

The integrin receptor M{beta}2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix and trans-endothelial migration of leukocytes. Here we present the first atomic structure of the human M{beta}2 headpiece fragment in complex with the nanobody hCD11bNb1 determined at a resolution of 3.2 [A]. The receptor headpiece adopts the closed conformation expected to have low ligand affinity. The crystal structure advocates that in the R77H M variant associated with systemic lupus erythematosus, the modified allosteric coupling between ligand coupling and integrin outside-inside signalling is due to subtle conformational effects transmitted over 40 [A]. The nanobody binds to the I domain of the M subunit in an Mg2+ independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins argue that the nanobody acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of iC3b attempting to bind the M subunit. Surprisingly, the nanobody stimulates the interaction of cell-bound M{beta}2 with iC3b suggesting that it represents a novel high-affinity proteinaceous M{beta}2 specific agonist. We propose a model based on the conformational spectrum of the receptor to reconcile these conflicting observations regarding the functional consequences of hCD11bNb1 binding to M{beta}2. Furthermore, our data suggest that the iC3b-M{beta}2 complex may be more dynamic than predicted from the crystal structure of the core complex.

immunology↗

bioRxiv2020

Complement receptor 3 forms a compact high affinity complex with iC3b

Complement receptor 3 (CR3, also known as Mac-1, integrin M{beta}2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment iC3b and to a lesser extent C3dg. While the interaction between the iC3b thioester domain and the ligand binding CR3 M I-domain is structurally and functionally well characterized, the nature of additional CR3-iC3b interactions required for phagocytosis of complement opsonized objects remain obscure. Here we analyzed the interaction between iC3b and the 150 kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of CR3 for iC3b compared to 515 nM for the iC3b thioester domain. Small angle x-ray scattering analysis revealed that iC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, iC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the iC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the iC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by iC3b than by C3dg and pave the way for development of specific therapeutics for treatment of inflammatory and neurodegenerative diseases that do not interfere with recognition of non-complement CR3 ligands.

biochemistry↗