Biology subjects

Grotjahn, D. A.

Publications and source records attributed to Grotjahn, D. A..

15 recordsLinked to original sources

bioRxiv2026

Structural and Mechanistic Basis of FapB Accessory Functional Amyloid Assembly in Pseudomonas

Functional amyloid formation in Pseudomonas biofilms is coordinated by the six-gene Fap operon (fapA-fapF), which governs the production, secretion, and assembly of extracellular fibrils that reinforce the biofilm matrix. FapC, FapB, and FapE constitute the extracellular amyloid. FapC is the major fibrillar component, whereas FapB and FapE occur at lower abundance and other accessory Fap proteins regulate fibril composition, processing, and secretion. We recently determined the structure of FapC, a cross-b-solenoid major Pseudomonas extracellular fibril. The assembly mechanism and architecture of FapB, however, have remained undefined. Here, we elucidate the structural and mechanistic basis of FapB accessory amyloid, from an intrinsically disordered monomer to a cross-b-solenoid fibril. Using well-defined monomeric preparations, controlled fibrillation assays, NMR spectroscopy, cryo-EM, and structure prediction, we show that FapB efficiently self-assembles into structurally homogeneous helical fibrils. FapB fibrillation is highly sensitive to its initial oligomeric state and purity, as well as to environmental factors such as pH. Monomeric FapC does not measurably perturb the disordered conformational ensemble of FapB, whereas preformed FapB and FapC fibrils reciprocally cross-seed each other, revealing direct kinetic coupling between the two amyloid components. Finally, we present a 3.4A resolution cryo-EM structure of the FapB accessory fibril, revealing a compact cross-b-solenoid architecture closely related to that of FapC. Together, these findings establish FapB as an independently amyloidogenic, structurally defined component of the Fap system, provide structural basis for the first of accessory cross-b-solenoid fibril, and reveal a mechanistic framework for the post-secretion assembly of multicomponent extracellular functional amyloids in Pseudomonas biofilms.

microbiology↗

bioRxiv2026

Siphoviridae phage tails co-enrich with ex vivo amyloids

Amyloid fibrils extracted from patient tissue are enriched by physicochemical fractionation methods and enzymatic tissue digestion with collagenase, resulting in a mixture of sedimented complexes. Reprocessing of an ex vivo lysozyme amyloid dataset revealed non-amyloid tubular assemblies, which we reconstructed to 3.5 angstrom. Sequence-agnostic model building with CryoAtom and fold search with FoldSeek identified a phage tail tube fold. Testing the crude Clostridium histolyticum collagenase used for tissue digestion, negative-stain TEM and LC-MS/MS showed proteins of an F-type tailocin, allowing us to assign the helical reconstruction to a major tail tube protein (TTP) of C. histolyticum. We also observed tailocin contamination in cardiac light-chain amyloid extracts, following the same collagenase treatment. Overall, we identify tailocins as recurrent reagent-borne contaminants in collagenase-treated ex vivo amyloid preparations and propose strategies to detect and avoid such artefacts in single-particle cryo-EM by optimizing the experimental design and data annotation towards a database of common contaminants.

biophysics↗

bioRxiv2026

Cathepsin-dependent amyloid formation drives mechanical rupture of lysosomal membranes

Lysosomal membrane integrity is essential for cellular homeostasis, and its failure drives lysosomal storage disorders (LSD) and neurodegeneration. The dipeptide L-leucyl-L-leucine methyl ester (LLOMe) is widely used to model lysosomal damage, yet its mechanism remains poorly understood. The prevailing view holds that LLOMe polymerizes into membrane-permeabilizing peptide chains within the lysosomal lumen. Using cryo-electron tomography in cultured cells and primary neurons, we visualized the structural basis of LLOMe-induced lysosomal damage. We reveal that LLOMe forms amyloid structures within lysosomes that directly interact with and rupture the limiting membrane through mechanical stress. In vitro reconstitution confirms this amyloid-mediated mechanism. These findings establish a structural paradigm for lysosomal membrane disruption and provide insights into how disease-relevant protein aggregates, implicated in neurodegeneration and LSD, may compromise lysosomal integrity.

cell biology↗

bioRxiv2025

Prohibitin complexes associate with unique membrane microdomains in cells

Prohibitins are multi-subunit protein complexes implicated in a variety of roles in mitochondria, including maintaining cristae architecture, regulating lipid metabolism, and mediating protein quality control. Prohibitins form large, ring-like structures within the mitochondrial inner membrane, and it has been proposed that this unique architecture enables them to organize distinct membrane environments that support their diverse functions. However, the molecular mechanisms driving these structure-function relationships within the native cellular context remain poorly defined. In this work, we combine cellular cryo-electron tomography, subtomogram averaging, and Surface Morphometrics analysis to systematically characterize the structure and local membrane environment of prohibitin complexes in mouse embryonic fibroblast (MEF) cells. We show that prohibitins are enriched in the inner boundary membrane subdomain of the inner mitochondrial membrane. Moreover, we find that a subset of prohibitin complexes exhibits a unique association with a matrix-facing density that resembles an m-AAA protease. Further, we show that prohibitins consistently localize to regions of reduced lipid bilayer thickness across all inner mitochondrial membrane subdomains, suggesting that they locally remodel the membrane to support their function. Together, these findings provide direct structural evidence that prohibitin complexes are associated with unique membrane microdomains in cells and establish a framework for uncovering how the local cellular context enables prohibitin function.

cell biology↗

bioRxiv2025

RobPicker: A Meta Learning Framework for Robust Identification of Macromolecules in Cryo-Electron Tomograms

Accurate particle picking of macromolecules of all kinds, shapes, and sizes in cryogenic electron tomograms (cryo-ET) is critical for understanding the molecular architecture of biological systems in their native state. Current deep learning methods have shown potential in identifying macromolecules from tomograms, but they are vulnerable to issues of noise in the training dataset obtained through human or automatic labeling and imbalanced distribution of macromolecule species. To address these limitations, we developed RobPicker, a meta-learning framework that effectively mitigates these issues by automatically learning deep neural networks to correct label errors and give greater emphasis to underrepresented macromolecule species. In evaluations across diverse cryo-ET datasets with noisy labels and imbalanced species distributions, RobPicker substantially outperforms state-of-the-art methods, particularly in identifying small and rare macromolecules. The efficiency and robustness of RobPicker can also be used for rapid fine-tuning of the tilt-series alignment, leading to improved tomogram reconstruction and enabling high-resolution cellular structural biology analysis.

bioinformatics↗

bioRxiv2024

Cryo-electron tomography reveals coupled flavivirus replication, budding and maturation

Flaviviruses replicate their genomes in replication organelles (ROs) formed as bud-like invaginations on the endoplasmic reticulum (ER) membrane, which also functions as the site for virion assembly. While this localization is well established, it is not known to what extent viral membrane remodeling, genome replication, virion assembly, and maturation are coordinated. Here, we imaged tick-borne flavivirus replication in human cells using cryo-electron tomography. We find that the RO membrane bud is shaped by a combination of a curvature-establishing coat and the pressure from intraluminal template RNA. A protein complex at the RO base extends to an adjacent membrane, where immature virions bud. Naturally occurring furin site variants determine whether virions mature in the immediate vicinity of ROs. We further visualize replication in mouse brain tissue by cryo-electron tomography. Taken together, these findings reveal a close spatial coupling of flavivirus genome replication, budding, and maturation.

microbiology↗

bioRxiv2024

Multi-Modal Large Language Model Enables All-Purpose Prediction of Drug Mechanisms and Properties

Accurately predicting the mechanisms and properties of potential drug molecules is essential for advancing drug discovery. However, traditional methods often require the development of specialized models for each specific prediction task, resulting in inefficiencies in both model training and integration into work-flows. Moreover, these approaches are typically limited to predicting pharmaceutical attributes represented as discrete categories, and struggle with predicting complex attributes that are best described in free-form texts. To address these challenges, we introduce DrugChat, a multi-modal large language model (LLM) designed to provide comprehensive predictions of molecule mechanisms and properties within a unified framework. DrugChat analyzes the structure of an input molecule along with users queries to generate comprehensive, free-form predictions on drug indications, pharmacodynamics, and mechanisms of action. Moreover, DrugChat supports multi-turn dialogues with users, facilitating interactive and in-depth exploration of the same molecule. Our extensive evaluation, including assessments by human experts, demonstrates that DrugChat significantly outperforms GPT-4 and other leading LLMs in generating accurate free-form predictions, and exceeds state-of-the-art specialized prediction models.

pharmacology and toxicology↗

bioRxiv2024

Multi-Modal Large Language Model Enables Protein Function Prediction

Predicting the functions of proteins can greatly accelerate biological discovery and applications, where deep learning methods have recently shown great potential. However, these methods predominantly predict protein functions as discrete categories, which fails to capture the nuanced and complex nature of protein functions. Furthermore, existing methods require the development of separate models for each prediction task, a process that can be both resource-heavy and time-consuming. Here, we present ProteinChat, a versatile, multi-modal large language model that takes a proteins amino acid sequence as input and generates comprehensive narratives describing its function. ProteinChat is trained using over 1,500,000 (protein, prompt, answer) triplets curated from the Swiss-Prot dataset, covering diverse functions. This novel model can universally predict a wide range of protein functions, all within a single, unified framework. Furthermore, ProteinChat supports interactive dialogues with human users, allowing for iterative refinement of predictions and deeper exploration of protein functions. Our experimental results, evaluated through both human expert assessment and automated metrics, demonstrate that ProteinChat outperforms general-purpose LLMs like GPT-4, one of the flagship LLMs, by over ten-fold. In addition, ProteinChat exceeds or matches the performance of task-specific prediction models.

bioinformatics↗

bioRxiv2024

Cytoplasmic ribosomes on mitochondria alter the local membrane environment for protein import

Most of the mitochondria proteome is nuclear-encoded, synthesized by cytoplasmic ribosomes, and targeted to mitochondria post-translationally. However, a subset of mitochondrial-targeted proteins is imported co-translationally, although the molecular mechanisms governing this process remain unclear. We employ cellular cryo-electron tomography to visualize interactions between cytoplasmic ribosomes and mitochondria in Saccharomyces cerevisiae. We use surface morphometrics tools to identify a subset of ribosomes optimally oriented on mitochondrial membranes for protein import. This allows us to establish the first subtomogram average structure of a cytoplasmic ribosome on the surface of the mitochondria in the native cellular context, which showed three distinct connections with the outer mitochondrial membrane surrounding the peptide exit tunnel. Further, this analysis demonstrated that cytoplasmic ribosomes primed for mitochondrial protein import cluster on the outer mitochondrial membrane at sites of local constrictions of the outer and inner mitochondrial membrane. Overall, our study reveals the architecture and the spatial organization of cytoplasmic ribosomes at the mitochondrial surface, providing a native cellular context to define the mechanisms that mediate efficient mitochondrial co-translational protein import. SUMMARYChang et al. present a membrane-guided approach for identifying a subset of cytoplasmic ribosomes oriented for protein import on the mitochondrial surface in Saccharomyces cerevisiae using cryo-electron tomography. They show that ribosomes cluster, make multiple contacts with, and induce local changes to the mitochondrial membrane ultrastructure at import sites.

cell biology↗

bioRxiv2023

Reentrant DNA shells tune polyphosphate condensate size

The ancient, inorganic biopolymer polyphosphate (polyP) occurs in all three domains of life and affects myriad cellular processes. An intriguing feature of polyP is its frequent proximity to chromatin, and in the case of many bacteria, its occurrence in the form of magnesium-enriched condensates embedded in the nucleoid, particularly in response to stress. The physical basis of the interaction between polyP and DNA, two fundamental anionic biopolymers, and the resulting effects on the organization of both the nucleoid and polyP condensates remain poorly understood. Given the essential role of magnesium ions in the coordination of polymeric phosphate species, we hypothesized that a minimal system of polyP, magnesium ions, and DNA (polyP-Mg2+-DNA) would capture key features of the interplay between the condensates and bacterial chromatin. We find that DNA can profoundly affect polyP-Mg2+ coacervation even at concentrations several orders of magnitude lower than found in the cell. The DNA forms shells around polyP-Mg2+ condensates and these shells show reentrant behavior, primarily forming in the concentration range close to polyP-Mg2+ charge neutralization. This surface association tunes both condensate size and DNA morphology in a manner dependent on DNA properties, including length and concentration. Our work identifies three components that could form the basis of a central and tunable interaction hub that interfaces with cellular interactors. These studies will inform future efforts to understand the basis of polyP granule composition and consolidation, as well as the potential capacity of these mesoscale assemblies to remodel chromatin in response to diverse stressors at different length and time scales.

biophysics↗

bioRxiv2022

Endosomal removal and disposal of dysfunctional, immunostimulatory mitochondrial DNA

Maternally inherited mitochondrial DNA (mtDNA) encodes essential subunits of the mitochondrial oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation1. This function of mtDNA contributes to antiviral resistance, but unfortunately also causes pathogenic inflammation in many disease contexts2. Cells experiencing mtDNA stress due to depletion of the mtDNA-packaging protein, Transcription Factor A, Mitochondrial (TFAM), or HSV-1 infection exhibit elongated mitochondria, mtDNA depletion, enlargement of nucleoids (mtDNA-protein complexes), and activation of cGAS/STING innate immune signaling via mtDNA released into the cytoplasm3. However, the relationships between altered mitochondrial dynamics and mtDNA-mediated activation of the cGAS-STING pathway remain unclear. Here, we show that entire enlarged nucleoids are released from mitochondria that remain bound to TFAM and colocalize with cGAS. These nucleoids arise at sites of mtDNA replication due to a block in mitochondrial fission at a stage when endoplasmic reticulum (ER) actin polymerization would normally commence, which we propose is a fission checkpoint to ensure that mtDNA has completed replication and is competent for segregation into daughter mitochondria. Released nucleoids also colocalize with the early endosomal marker RAB5 as well as the late endosomal marker RAB7 in TFAM-deficient cells and in response to mtDNA stress caused by the HSV-1 UL12.5 protein. Loss of RAB7 increases interferon stimulated gene (ISG) expression. Thus, we propose that defects in mtDNA replication and/or segregation enact a late mitochondrial fission checkpoint that, if persistent, leads to selective removal of dysfunctional nucleoids by a mitochondrial-endosomal pathway. Early steps in this pathway are prone to mtDNA release and cGAS-STING activation, but the immunostimulatory mtDNA is ultimately disposed of through a mechanism involving RAB7-containing late endosomes to prevent excessive innate immune signaling. This mtDNA quality control pathway might represent a therapeutic target to prevent mtDNA-mediated inflammation and associated pathology.

cell biology↗

bioRxiv2022

A surface morphometrics toolkit to quantify organellar membrane ultrastructure using cryo-electron tomography

Cellular cryo-electron tomography (cryo-ET) enables 3-dimensional reconstructions of organelles in their native cellular environment at subnanometer resolution. However, quantifying ultrastructural features of pleomorphic organelles in three dimensions is challenging, as is defining the significance of observed changes induced by specific cellular perturbations. To address this challenge, we established a semi-automated workflow to segment organellar membranes and reconstruct their underlying surface geometry in cryo-ET. To complement this workflow, we developed an open source suite of ultrastructural quantifications, integrated into a single pipeline called the surface morphometrics toolkit. This toolkit allows detailed mapping of spacing, curvature, and orientation onto reconstructed membrane meshes, highlighting subtle organellar features that are challenging to detect in three dimensions and allowing for statistical comparison across many organelles. To demonstrate the advantages of this approach, we combine cryo-ET with cryo-fluorescence microscopy to correlate bulk mitochondrial network morphology (i.e., elongated versus fragmented) with membrane ultrastructure of individual mitochondria in the presence and absence of endoplasmic reticulum (ER) stress. Using our toolkit, we demonstrate ER stress promotes adaptive remodeling of ultrastructural features of mitochondria including spacing between the inner and outer membranes, local curvature of the inner membrane, and spacing between mitochondrial cristae. We show that differences in membrane ultrastructure correlate to mitochondrial network morphologies, suggesting that these two remodeling events are coupled. Our toolkit offers opportunities for quantifying changes in organellar architecture on a single-cell level using cryo-ET, opening new opportunities to define changes in ultrastructural features induced by diverse types of cellular perturbations.

cell biology↗

bioRxiv2021

Nanoscale details of mitochondrial fission revealed by cryo-electron tomography

Mitochondrial fission is required for proper segregation during cell division, quality control, and cellular homeostasis (metabolism and energy production). Despite its importance, models of the process remain speculative. Here we apply cryogenic electron tomography to image the nanoscale architecture of mitochondrial fission in mammalian cells. We find that constriction of the inner and outer membranes is coordinated, suggesting that force on both membranes is applied externally. While we observe ER at constriction sites, it did not encircle constrictions. Instead, we find long bundles of both unbranched actin and septin filaments enriched at constrictions. Actin bundles align with the central region of division bridges and septin bundles with the necks on either side. Septin bundles appear to guide microtubules to constriction sites, suggesting, along with autolysosomes observed in the vicinity, a pathway for mitophagy. Together, our results rule out several existing models for mitochondrial fission and provide empirical parameters to inform the development of realistic coarse-grained models in the future.

cell biology↗

bioRxiv2021

A case for glycerol as an acceptable additive to cryoEM samples

Buffer composition and sample preparation guidelines for cryo-electron microscopy are geared toward maximizing imaging contrast and reducing electron beam-induced motion. These pursuits often involve the minimization or complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, we show that inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by a [~]2.3 [A] reconstruction of mouse apoferritin ([~]500 kDa) and a [~]3.3 [A] reconstruction of rabbit muscle aldolase ([~]160 kDa) in presence of 20% v/v glycerol. While we found that generating thin ice that is amenable for high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion nor an inability to pick particles. Overall, our findings indicate glycerol should not be discounted as a cryoEM sample buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.

biophysics↗

bioRxiv2017

Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility

A key player in the intracellular trafficking network is cytoplasmic dynein, a protein complex that transports molecular cargo along microtubule tracks. It has been shown that vertebrate dyneins movement becomes strikingly enhanced upon interacting with a cofactor named dynactin and one of several cargo-adapters, such as BicaudalD2. However, the mechanisms responsible for this increase in transport efficiency are not well understood, largely due to a lack of structural information. We used cryo-electron tomography to visualize the first 3-dimensional structure of the intact dynein-dynactin complex bound to microtubules. Our structure reveals that the dynactin-cargo-adapter complex recruits and binds to two dimeric cytoplasmic dyneins. Interestingly, the dynein motor organization closely resembles that of axonemal dynein, suggesting that cytoplasmic dynein and axonemal dyneins may utilize similar mechanisms to coordinate multiple motors. We propose that grouping dyneins onto a single dynactin scaffold promotes collective force production as well as unidirectional processive motility. These findings provide a structural platform that facilitates a deeper biochemical and biophysical understanding of dynein regulation and cellular transport.

cell biology↗