Biology subjects

Jakobi, A. J.

Publications and source records attributed to Jakobi, A. J..

16 recordsLinked to original sources

bioRxiv2026

State-selective nanobodies to probe Guanylate-binding protein coat assembly and function

Cell-autonomous immunity against gram-negative bacterial pathogens relies on Guanylate-binding proteins (GBPs). GBPs are interferon-inducible guanosine triphosphate hydrolases that coat cytosolic bacteria and promote non-canonical inflammasome activation. GBP coat assembly is initiated by GBP1, which binds bacterial lipopolysaccharide and recruits further paralogs in a hierarchical and nucleotide-dependent manner. Dissecting the contributions of individual GBPs has remained challenging because the seven human paralogs are structurally similar and undergo nucleotide-driven conformational changes that are difficult to resolve in cellular and biochemical experiments. Here, we developed a nanobody toolbox for the state-selective probing of human GBP coat assembly. We identify eight nanobodies with distinct paralog, domain, and conformational specificities and define their binding profiles and epitopes using complementary biophysical assays and cryogenic electron microscopy. We further demonstrate the utility of selected nanobodies by demonstrating their functionality in membrane reconstitution assays and in infected cells. A dimer-disrupting nanobody blocks progression of GBP-coat assembly from soluble GBP1 assemblies to LPS-associated coats, suggesting a stage-specific structural transition during coat formation. In contrast, a dimer selective nanobody binds assembled coats without measurably perturbing their organisation and is robustly recruited to GBP1-positive bacteria in infected cells, indicating that this dimer interface remains intact and accessible in coat-associated GBP1. Together, these nanobodies establish a set of state-selective reagents to distinguish, perturb, and probe GBP assemblies in structural and cellular studies.

biophysics↗

bioRxiv2026

Structural conservation and expanded functionality of hyper-stable human serum albumin variants

Human serum albumin (hSA) is the most abundant protein in human plasma, and its pharmacological properties, such as long plasma half-life mediated by the neonatal Fc receptor (FcRn) and its ability to bind endogenous and exogenous molecules, make it attractive for biotechnological applications. Currently, most wild type (WT) SAs are derived from human or bovine serum or produced in yeast and mammalian cells. Although well established, these methods are costly, difficult to reproduce, and not environmentally sustainable. Building on a previous study to design highly mutated hSA sequences, we extend the validation through an in-depth analysis of three engineered hSA variants; hSA1, hSA2, and hSA3, containing 16, 25, or 73 amino acid substitutions, respectively. These variants were designed for enhanced solubility, stability, and expression in Escherichia coli. All three variants showed low- micromolar affinities for hFcRn at pH 5.5, and negligible binding at pH 7.4. In a human endothelial cell-based recycling assay (HERA), the engineered hSA variants were recycled by hFcRn to the same extent as hSA isolated from serum. Exploring the properties of canonical drug-binding sites, warfarin affinity was comparable to WT hSA, whereas ibuprofen binding differed. Complementary cytotoxicity assays on human macrophages confirmed negligible toxicity and biocompatibility. A cryo-electron microscopy structure of hSA3 revealed that, despite extensive engineering, the native heart-shape of hSA, folding of domains, and its open conformation were preserved. These findings validate the structural integrity and functional adaptability of engineered hSA variants, underscoring their potential as versatile, animal-free solutions for next-generation therapeutics and biotechnological applications.

biochemistry↗

bioRxiv2026

Interactive segmentation of membrane and membrane mimic densities in cryo-EM maps

Contextual density arising from biological membranes or membrane mimics such as detergent micelles or lipid nanodiscs, is ubiquitous in cryo-EM maps of integral and peripheral membrane proteins, or membrane-associated complexes, from single-particle analysis and subtomogram averaging. Here, we describe SURFER, a lightweight, GPU-accelerated extension for UCSF ChimeraX that enables rapid semantic differentiation of membrane or membrane mimic density from macromolecular signal. SURFER operates on unfiltered maps to automatically generate segmentation masks that can be flexibly applied to any aligned target map, including reference volumes for local refinement or post-processed maps optimised for visualisation and interpretation. By allowing contextual density to be selectively toggled or subtracted, SURFER enables direct interactive comparison between representations with and without membrane-related features to facilitate consistent analysis and reporting of membrane context in cryo-EM structures. An interactive graphical interface within ChimeraX supports convenient threshold adjustment, connectivity filtering, and immediate visual evaluation of the resulting segmentation.

biophysics↗

bioRxiv2025

Confidence-guided cryo-EM map optimisation with LocScale-2.0

Cryogenic sample electron microscopy (cryo-EM) maps often display uneven quality, with high-resolution features coexisting alongside weak or poorly ordered regions. Such variation complicates structural interpretation, especially for heterogeneous macromolecular assemblies. Here, we present LocScale-2.0, a context-aware map optimisation framework that operates without prior knowledge of molecular structure or composition. By leveraging general expectations of electron scattering by biological macromolecules, it enhances local detail and connectivity while preserving weak but biologically relevant structural context. We further introduce LocScale-FEM, a Bayesian-approximate deep-learning approach that emulates this optimisation to generate feature-enhanced maps. LocScale-FEM provides voxel-wise confidence scores that give a statistically grounded measure of reliability, are sensitive to local phase error in the feature-enhanced map, and highlight regions where interpretation warrants caution. Selected examples illustrate how such maps can aid biological interpretation and how confidence-guided map optimisation can increase objectivity in cryo-EM density analysis.

biophysics↗

bioRxiv2024

Thickness and quality controlled fabrication of fluorescence-targeted frozen-hydrated lamellae

Cryogenic focused ion beam (FIB) milling is essential for fabricating thin lamella-shaped samples out of frozen-hydrated cells for high-resolution structure determination. Structural information can only be resolved at high resolution if the lamella thickness is between 100 and 200 nm. While the lamella fabrication workflow has undergone significant improvements since its conception, quantitative, live feedback on lamella thickness and quality is still lacking. Taking advantage of a coincident light microscopy integrated into the FIB-SEM, we present three different strategies that together allow accurate, live control during lamella fabrication. First, we combine 4D-STEM with fluorescence microscope (FM) targeting to determine the lamella thickness. Second, with reflected light microscopy (RLM) we screen target sites for ice contamination and monitor lamella thickness and integrity of the protective Pt coating during FIB milling. Third, we exploit thin-film interference to obtain fine-grained feedback on thickness uniformity below 500 nm. We finally present a full workflow for fluorescence-targeted and quality controlled fabrication of frozen-hydrated lamellae, benchmarked with excellent agreement to energy filtered transmision electron microscopy (EFTEM) measurements and reconstructed tomograms obtained with electron cryo-tomography.

biophysics↗

bioRxiv2024

Roodmus: A toolkit for benchmarking heterogeneous electron cryo-microscopy reconstructions

Conformational heterogeneity of biological macromolecules is a challenge in single particle averaging (SPA). Current standard practice is to employ classification and filtering methods which may allow a discrete number of conformational states to be reconstructed. However, the conformation space accessible to these molecules is continuous and therefore explored incompletely by a small number of discrete classes. Recently developed heterogeneous reconstruction algorithms (HRAs) to analyse continuous heterogeneity rely on machine learning methods employing low-dimensional latent space representations. The non-linear nature of many of these methods pose challenges to their validation and interpretation, and to identifying functionally relevant conformational trajectories. We believe these methods would benefit from in-depth benchmarking using high quality synthetic data and concomitant ground truth information. Here we present a framework for the simulation and subsequent analysis with respect to ground-truth of cryo-EM micrographs containing particles whose conformational heterogeneity is sourced from molecular dynamics simulations. This synthetic data can then be processed as if it were experimental data allowing aspects of standard SPA workflows, as well as heterogeneous reconstruction methods, to be compared with known ground-truth using available utilities. We will demonstrate the simulation and analysis of several such datasets and present an initial investigation into HRAs.

biophysics↗

bioRxiv2024

Snapshots of Pseudomonas aeruginosa SOS response activation complex reveal structural prerequisites for LexA engagement and cleavage

Antimicrobial resistance represents a major threat to human health and Pseudomonas aeruginosa stands out among the pathogens responsible for this emergency. The SOS response to DNA damage plays a pivotal role in bacterial evolution, driving the development of resistance mechanisms and influencing the adaptability of bacterial populations to challenging environments, particularly in the context of antibiotic exposure. Recombinase A (RecA) and the transcriptional repressor LexA are the key players that orchestrate this process, determining either the silencing or the active transcription of the genes under their control. By integrating state-of-the-art structural approaches with binding and functional assays in vitro, we elucidated the molecular events governing the SOS response activation in P. aeruginosa, focusing on the RecA-LexA interaction. Our findings identify the conserved determinants and strength of the interactions that let RecA trigger the autocleavage and inactivation of the LexA repressor. These results provide the groundwork for designing novel antimicrobial strategies and for exploring the potential translation of Escherichia coli-derived approaches, to address the health-threatening implications of bacterial infections. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/585941v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@2fe1e4org.highwire.dtl.DTLVardef@19741e1org.highwire.dtl.DTLVardef@1664ddborg.highwire.dtl.DTLVardef@18195cb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Structural basis of membrane targeting and coatomer assembly by human GBP1

Guanylate-Binding Proteins (GBPs) are interferon-inducible guanosine triphosphate hydrolases (GTPases) that mediate immune effector functions against intracellular pathogens. A key step for the antimicrobial activity of GBPs is the formation of homo- and heterooligomeric complexes on the membrane of pathogen-associated compartments or cytosolinvasive bacteria. Similar to other large GTPases of the dynamin family, oligomerisation and membrane association of GBPs depend on their GTPase activity. How nucleotide binding and hydrolysis prime GBPs for membrane targeting and coatomer formation remains unclear. Here, we report the cryo-EM structure of the full-length human GBP1 dimer in its guanine nucleotide-bound state and resolve the molecular ultrastructure of GBP1 coatomer assemblies on liposomes and bacterial lipopolysaccharide membranes. We show how nucleotide binding promotes large-scale conformational changes of the middle and GTPase effector domains that expose the isoprenylated carboxyl-terminus for association with lipid membranes. Our structure reveals how the -helical stalks of the middle domain form a parallel arrangement firmly held in a unique cross-over arrangement by intermolecular contacts between adjacent monomers. This conformation is critical for GBP1 dimers to assemble into densely packed coatomers on target membranes. The extended -helix of the effector domain is flexible and permits intercalation into the dense lipopolysaccharide layer on the outer membrane of gram-negative bacterial pathogens. We show that nucleotide-dependent oligomerisation and GTP hydrolysis yield GBP1 membrane scaffolds with contractile abilities that promote the formation of tubular membrane protrusions and membrane fragmentation. Collectively, our data provide a structural and mechanistic framework for interrogating the molecular basis for GBP1 effector functions in intracellular immunity.

biophysics↗

bioRxiv2023

THE RIBOREGULATION MECHANISM OF HUMAN SERINE HYDROXYMETHYLTRANSFERASE IS ROOTED IN AN ALLOSTERIC SWITCH

RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of these being characterized on structural grounds. Here we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzymes reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in the evolution of eukaryotic SHMT1 and the compartmentalization of one-carbon metabolism. The findings also provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

biochemistry↗

bioRxiv2022

Particle fusion of Single Molecule Localization Microscopy data reveals dimer structure of Nup96 in Nuclear Pore Complex

Single molecule localization microscopy offers nowadays resolution nearly down to the molecular level with specific molecular labelling, thereby being a promising tool for structural biology. In practice, however, the actual value to this field is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles equivalent to cryo-EM single-particle analysis. In this analysis, we present particle averaging of fluorescently labelled Nup96 in the Nuclear Pore Complex followed by data analysis to show that Nup96 occurs as a dimer with in total 32 copies per pore. We use Artificial Intelligence assisted modeling in Alphafold to extend the existing cryo-EM model of Nup96 to accurately pinpoint the positions of the fluorescent labels and show the accuracy of the match between fluorescent and cryo-EM data to be better than 3 nm in-plane and 5 nm out-of-plane.

cell biology↗

bioRxiv2022

A cryogenic, coincident fluorescence, electron and ion beam microscope

Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualisation and structure determination of macromolecular complexes at sub-nanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in-situ fluoresence microscopy-guided FIB fabrication of a frozen-hydrated lamella to solve this problem: we built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope (FIB-SEM). We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

molecular biology↗

bioRxiv2022

Cryo-EM structure of gas vesicles for buoyancy-controlled motility

Gas vesicles allow a diverse group of bacteria and archaea to move in the water column by controlling their buoyancy (1). These gas-filled cellular nanocompartments are formed by up to micrometers long protein shells that are permeable only to gas. The molecular basis of their unique properties and mechanism of assembly remains unknown. Here, we solve the 3.2 [A] cryo-EM structure of the B.megaterium gas vesicle shell made from the structural protein GvpA that self-assembles into hollow helical cylinders closed off by cone-shaped tips. Remarkably, the unique fold adopted by GvpA generates a corrugated cylinder surface typically found in force-bearing thin-walled structures. We identified pores in the vesicle wall that enable gas molecules to freely diffuse in and out of the GV shell, while the exceptionally hydrophobic interior surface effectively repels water. Our results show that gas vesicles consist of two helical half-shells connected through a unique arrangement of GvpA monomers, suggesting a mechanism of gas vesicle biogenesis. Comparative structural analysis confirms the evolutionary conservation of gas vesicle assemblies and reveals molecular details of how the secondary structural protein GvpC reinforces the GvpA shell. Our findings provide a structural framework that will further research into the biology of gas vesicles, and enable rational molecular engineering to harness their unique properties for acoustic imaging (2, 3).

biophysics↗

bioRxiv2021

Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus ends

Growing microtubule ends provide platforms for the accumulation of plus-end tracking proteins that organize into comets of mixed protein composition. Using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions appear to facilitate the non-stoichiometric accumulation of cargo Tip1. We dissected the contribution of two disordered protein regions in Mal3 and found that both are required for the ability to form droplets and Tip1 accumulation, while autonomous Mal3 comet formation only requires one of them. Using theoretical modeling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends.

cell biology↗

bioRxiv2021

Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes

Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.

biophysics↗

bioRxiv2019

Structural basis of p62/SQSTM1 helical filaments, their presence in p62 bodies and role in cargo recognition in the cell

p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We determined four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of phase-separated p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo recognition and are an integral part of phase separated p62 bodies.

molecular biology↗

bioRxiv2017

Iterative model-based density improvement yields better atomic structures from cryo-EM maps

Atomic models based on high-resolution density maps are the ultimate result of the cryo-EM structure determination process. Current cryo-EM model refinement procedures work with the experimental density map that remains constant throughout the process. Here, we introduce a general procedure that iteratively improves cryo-EM density maps based on prior knowledge of an atomic reference structure. The procedure optimizes contrast of cryo-EM densities by local amplitude scaling (LocScale) based on an atomic model without introducing model bias. We alternate the procedure with consecutive rounds of model refinement and tested it on four cryo-EM structures of TRPV1, {beta}-galactosidase, {gamma}-secretase and RNA polymerase III. We demonstrate that LocScale density improvement reveals previously undiscovered map features and improves the quality of atomic models. The presented approach enhances the interpretability of cryo-EM density maps and provides an implementation reminiscent of iterative density improvement as it is routinely employed in the refinement of X-ray crystallographic models.

biophysics↗