Biology subjects

Agard, D. A.

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

At least 19 recordsLinked to original sources

bioRxiv2026

IRE1α undergoes a dynamic filament assembly cycle governed by unfolded proteins and BiP.

The endoplasmic reticulum is a major hub of protein synthesis, modification and trafficking that relies on the Unfolded Protein Response (UPR) to balance its folding capacity. IRE1, the most conserved of the UPR sensors, undergoes a change in oligomeric state upon activation that propagates the stress signal from the ER lumen to its cytoplasmic kinase/RNase domains. Here we show that opposing inputs from unfolded proteins and the ER chaperone machinery govern a dynamic cycle of IRE1 assembly. A bona fide unfolded ER client directly binds the IRE1 lumenal domain (LD) and promotes higher-order oligomerization. Model peptides further reveal that full assembly into LD filaments depends on ligand length, sequence, and the IRE1 oligomerization interfaces. The ER Hsp70, BiP, counterbalances this process through two distinct mechanisms: i) an ATP-independent interaction via the BiP nucleotide-binding domain buffers early IRE1 assembly by engaging its oligomerization interface, whereas ii) ATP-regulated BiP/ERDJ machinery actively dismantles assembled filaments. Together, our findings reconcile direct unfolded protein recognition with BiP-mediated regulation to establish LD oligomerization as a point of integration through which unfolded protein load and chaperone availability dynamically regulate IRE1 assembly state and signalling.

biochemistry↗

bioRxiv2026

Structural and mechanistic analyses reveal collaborative regulation of HSF1 by the Hsp70-Hsp90 chaperone systems

Heat shock factor 1 (HSF1) is the master transcriptional regulator of cellular response to disrupted cytosolic protein homeostasis. Temperature change, oxidation, and other stresses drive the trimerization and activation of HSF1 to induce expression of molecular chaperones, such as heat shock proteins Hsp70 and Hsp90, which sit at the center of cellular proteostatic networks. In turn, the HSPs and co-chaperones regulate HSF1, but mechanistic details of this cycle remain largely unknown. We developed a FRET-based approach to simultaneously monitor HSF1 conformational change and oligomeric state throughout activation and inactivation. By reconstituting Hsp-HSF1 interactions in vitro, we find that monomerization of HSF1 resembles fibril disassembly through coordinated Hsp40-Hsp70 activity. We then used site-specific photocrosslinking to track HSF1 loading into Hsp90 complexes, Hsp90 cycling, and stress-induced shifts in Hsp90-HSF1 interactions. Whereas Hsp90 inhibitors force 'loading state' type Hsp90-HSF1 interactions, heat shock promotes faster Hsp90 cycling. In this reconstituted system, HSF1-Hsp90 interactions are unexpectedly strongly dependent on the co-chaperone HOP, in contrast to canonical Hsp90 clients. Combining cryo-EM structures of Hsp90-HSF1 loading state and maturation state complexes with crosslinking mass spectroscopy and biophysical experiments, we show that Hsp90 holds HSF1 in a pre-activated, extended monomer state that is primed for trimerization. We propose this state both enhances responsivity but also promotes cytoplasmic-nuclear shuttling through exposure of the NLS. Notably, Hsp90-bound HSF1 can trimerize and bind DNA, placing it on-pathway for transcriptional activation. Together, our results unify previously contradictory view on Hsp90's role in HSF1 regulation. The integrated combination of in vitro reconstitution, photocrosslinking, cryoEM and MS is an exciting new paradigm for the study of many dynamic systems including other complex proteostasis components.

biochemistry↗

bioRxiv2026

High Accuracy Fluorescence Guided Focused Ion Beam Milling

Cryo-electron tomography (cryo-ET) is a powerful approach for visualizing macromolecular structures directly within cells, but its broader application is limited by the difficulty of reliably targeting specific structures during sample preparation. In particular, capturing small or rare objects within cryo-focused ion beam (cryo-FIB) milled lamellae remains a major bottleneck. Here, we present two fluorescence-guided cryo-FIB milling workflows that overcome key sources of targeting error and enable routine capture of structures across a broad range of spatial scales. For larger targets (>500 nm), we present an improved registration-based strategy combining FIB-milled fiducials with depth correction for refractive index mismatch. For smaller targets (150-500 nm), we implement real-time fluorescence guided milling on a commercially available cryo-FIB-SEM platform enabling consistent recovery of small, low-copy organelles such as centrosomes and primary cilia. Both workflows are implemented in a new open-source package fibsemOS and together expand the range of cellular structures accessible to cryo-ET and establish a broadly deployable framework for targeted in situ structural biology.

cell biology↗

bioRxiv2026

Double-membrane-spanning RNA export pores are a conserved feature in nidovirus replication organelles

Upon infection, arteriviruses, coronaviruses, and other nidoviruses transform endoplasmic reticulum membranes into viral replication organelles. These include large numbers of double-membrane vesicles (DMVs) whose interior is considered the primary site of viral RNA synthesis. Early studies characterized nidovirus DMVs as sealed compartments, leaving it unclear how newly synthesized viral RNA could be exported to the cytosol. The discovery of DMV-spanning pore complexes in coronavirus-infected cells provided a plausible solution for this topological challenge. However, their structural organization, functional features, and evolutionary conservation across the nidovirus order, have remained unclear. Here, we investigated the macromolecular architecture of DMVs induced by two prototypic arteriviruses using cellular cryo-electron tomography. Despite the substantial evolutionary distance separating arteriviruses and coronaviruses, we observed DMV-spanning pore complexes with striking structural similarities to those previously described in coronaviruses. These pores appear to facilitate both export and encapsidation of viral RNA. In the absence of viral RNA synthesis, ectopic expression of the arterivirus transmembrane nonstructural proteins nsp2 and nsp3 sufficed to induce the formation of pore-containing DMVs. Together, our findings reveal the conservation of key structural features of DMV pores across two distantly related nidovirus families and support a central role for these pores in nidovirus replication.

microbiology↗

bioRxiv2025

Cathepsin C-Catalyzed Ligation Generates Intralysosomal Amyloid Fibrils from Dipeptide Esters

The lysosome is a major catabolic organelle responsible for the breakdown of both intra- and extracellular substrates1,2. Lysosomal membrane damage mediated by pathologic amyloid fibrils is an area of recent focus3-6. The dipeptide ester LLOMe is typically employed to model lysosomal membrane damage7-11; however its mechanism of membranolysis was previously incompletely understood. Here, in vitro and cell-based analyses, and cryo-electron microscopy and tomography studies reveal LLOMe-derived oligopeptides generated by the lysosomal protease Cathepsin C assemble into cross-{beta}-sheet amyloid fibrils within the lysosome. Additionally, we report lysosome membrane damage triggers the broadly nonspecific dipeptidyl ligase activity of Cathepsin C, facilitating the tagging of proximal proteins within the damaged lysosome lumen with a click chemistry handle: to our knowledge, the first reported localized proximity labeling approach exploiting a fully endogenous, non-engineered enzyme. While Cathepsin C ligase activity has been demonstrated in vitro12,13, our observations of dipeptidyl ligation onto proximal proteins in cells suggests an unexplored role of Cathepsin C in lysosomal biology and broadly exemplifies how other endogenous enzymes might be similarly exploited for proximity labeling. Altogether our results unveil two mechanisms by which dipeptide esters perturb lysosomal homeostasis and provide a roadmap for their utilization toward targeted studies of the lysosome.

biochemistry↗

bioRxiv2025

Lessons learned from a Kaggle challenge for particle picking in cryo-electron tomography

The difficulty of particle picking in cryo-electron tomography remains a barrier to routine in situ structure determination. Machine learning is well-suited to overcome this bottleneck with efficient algorithms that generalize across molecular species. To spur new algorithm development, we held a three-month Kaggle challenge that tasked contestants with annotating five molecular species across hundreds of experimental tomograms. This competition successfully engaged >1000 participants from diverse fields and delivered particle pickers that outperformed existing state-of-the-art. Systematic comparisons of the contestants submissions revealed the tolerance of subtomogram averaging to moderate but not severe over-picking and underscored the need for more robust measures of annotation quality. The winning models also highlighted the importance of data augmentation to overcome limited training data. All competition tomograms along with the ground truth and winning teams annotations have been released on the CryoET Data Portal as a resource to benchmark current and future tools for particle picking.

cell biology↗

bioRxiv2025

Hsp90-dependent triage of the glucocorticoid receptor via the CHIP E3 ubiquitin ligase

The molecular chaperone Hsp90 is required to ensure the proper folding and activation of approximately 10% of the proteome throughout their cellular lifetime1,2. Given that Hsp90 recognizes and preferentially interacts with partially unfolded client states, it is well-poised to promote the degradation of misfolded clients and clients past the point of re-foldability3,4,5. Long- standing phenomenological data suggest that Hsp90 could directly present a client protein to an E3 ubiquitin ligase, in this way mediating degradative ubiquitylation of the client. However, this has yet to be directly demonstrated for any client. To assess this mechanism for the model Hsp90 client the glucocorticoid receptor (GR), a ligand-dependent transcription factor, we reconstituted in vitro the Hsp90-dependent folding cycle for GR6, in the presence of the E3 ligase CHIP. Remarkably, we discovered that CHIP binds to and efficiently ubiquitylates GR as presented by a previously characterized Hsp90-dependent pro-folding state called the GR-maturation state (Hsp90-p23-GR), in which GR has been visualized via cryo-EM to be folded in the native state and ligand-bound7. We determined a low-resolution ([~]14 [A]) cryo-EM reconstruction of the four- component Hsp90-p23-GR-CHIP complex, and furthermore resolved from the same dataset a 3.53 [A] reconstruction of a Hsp90-p23-GR complex in which, surprisingly, GR is partially disordered and ligand-free--in contrast with what we previously observed7, presumably as a consequence of interaction with CHIP. Our results suggest a mechanism by which Hsp90 can simultaneously partition a client between both folding and degradation outcomes and in this way directly mediate protein-triage decisions through its interactions with E3 ligases.

biochemistry↗

bioRxiv2025

AreTomoLive: Automated reconstruction of comprehensively-corrected and denoised cryo-electron tomograms in real-time and at high throughput

A high throughput processing pipeline that performs comprehensive corrections is needed to realize the full potential of cryo-electron tomography and subtomogram averaging. The fields fragmented software landscape remains a significant hurdle to this end. Here we present AreTomoLive, an automated real-time pipeline composed of two GPU-accelerated packages. The first, AreTomo3, streamlines tomographic alignment and reconstruction, with new features to fully account for sample geometry, locally correct the contrast transfer function, and curate data for downstream tasks. The second package, DenoisET, is a new implementation of the machine learning algorithm Noise2Noise and runs in parallel with AreTomo3 to perform contrast enhancement. To reduce barriers to routine use, AreTomoLive prioritizes automation: AreTomo3 autonomously pauses and reactivates processing depending on the status of data collection, while DenoisET algorithmically determines when to transition from training to inference. AreTomoLive endeavors to advance cryoET for in situ structural analysis with its comprehensive corrections and full automation.

cell biology↗

bioRxiv2025

Deconvolution to restore cryo-EM maps with anisotropic resolution

With technological advancements in recent years, single particle cryogenic electron microscopy (cryo-EM) has become a major methodology for structural biology. Structure determination by single particle cryo-EM is premised on randomly orientated particles embedded in a thin layer of vitreous ice to resolve high-resolution structure in all directions. In practice, preferentially distributed particle orientations and/or other imperfections in imaging and data processing deteriorate quality of obtained cryo-EM map. Here we present a deconvolution approach, named AR-Decon, that computationally improves the quality of cryo-EM maps. We tested and validated the procedure, compared its performance with that of machine learning based density modification method, and benchmarked its performance with a wide range of deposited maps. Our results show that AR-Decon is robust and is a generally applicable post-processing procedure for single particle cryo-EM.

biophysics↗

bioRxiv2024

The Structure of Cilium Inner Junctions Revealed by Electron Cryo-tomography

The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryo-tomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to "infiltrate" into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner junctions, underscoring its precise spatial regulation.

cell biology↗

bioRxiv2023

Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking

Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). To address this issue, we developed graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer. These grids protect complexes between the chromatin remodeler SNF2h and nucleosomes from the AWI and facilitated collection of high-quality micrographs of intact SNF2h-nucleosome complexes in the absence of crosslinking. The data yields maps ranging from 2.3 to 3 [A] in resolution. 3D variability analysis reveals nucleotide-state linked conformational changes in SNF2h bound to a nucleosome. In addition, the analysis provides structural evidence for asymmetric coordination between two SNF2h protomers acting on the same nucleosome. We envision these grids will enable similar detailed structural analyses for other enzyme-nucleosome complexes and possibly other protein-nucleic acid complexes in general.

biochemistry↗

bioRxiv2023

Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the Glucocorticoid Receptor

Hsp90 is an essential molecular chaperone responsible for the folding and activation of hundreds of client proteins, including the glucocorticoid receptor (GR)1-3. Previously, we revealed that GR ligand binding activity is inhibited by Hsp70 and restored by Hsp90, aided by co-chaperones4. We then presented cryo-EM structures mechanistically detailing how Hsp70 and Hsp90 remodel the conformation of GR to regulate ligand binding5,6. In vivo, GR-chaperone complexes are found associated with numerous Hsp90 co-chaperones, but the most enigmatic have been the immunophilins FKBP51 and FKBP52, which further regulate the activity of GR and other steroid receptors7-9. A molecular understanding of how FKBP51 and FKBP52 integrate with the GR chaperone cycle to differentially regulate GR activation in vivo is lacking due to difficulties reconstituting these interactions. Here, we present a 3.01 [A] cryo-EM structure of the GR:Hsp90:FKBP52 complex, revealing, for the first time, that FKBP52 directly binds to the folded, ligand-bound GR using three novel interfaces, each of which we demonstrate are critical for FKBP52-dependent potentiation of GR activity in vivo. In addition, we present a 3.23 [A] cryo-EM structure of the GR:Hsp90:FKBP51 complex, which, surprisingly, largely mimics the GR:Hsp90:FKBP52 structure. In both structures, FKBP51 and FKBP52 directly engage the folded GR and unexpectedly facilitate release of p23 through an allosteric mechanism. We also reveal that FKBP52, but not FKBP51, potentiates GR ligand binding in vitro, in a manner dependent on FKBP52-specific interactions. Altogether, we reveal how FKBP51 and FKBP52 integrate into the GR chaperone cycle to advance GR to the next stage of maturation and how FKBP51 and FKBP52 compete for GR:Hsp90 binding, leading to functional antagonism.

biochemistry↗

bioRxiv2022

De novo protein identification in mammalian sperm using high-resolution in situ cryo-electron tomography

Understanding molecular mechanisms of cellular pathways requires knowledge of the identities of participating proteins, their cellular localization and their 3D structures. Contemporary workflows typically require multiple techniques to identify target proteins, track their localization using fluorescence microscopy, followed by in vitro structure determination. To identify mammal-specific sperm proteins and understand their functions, we developed a visual proteomics workflow to directly address these challenges. Our in situ cryo-electron tomography and subtomogram averaging provided 6.0 [A] resolution reconstructions of axonemal microtubules and their associated proteins. The well-resolved secondary and tertiary structures allowed us to computationally match, in an unbiased manner, novel densities in our 3D reconstruction maps with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105 and SPACA9 as novel microtubule inner proteins that form an extensive network crosslinking the lumen of microtubule and existing proteins. Additional biochemical and mass spectrometry analyses helped validate potential candidates. The novel axonemal sperm structures identified by this approach form an extensive interaction network within the lumen of microtubules, suggesting they have a role in the mechanical and elastic properties of the microtubule filaments required for the vigorous beating motions of flagella.

biophysics↗

bioRxiv2022

A family of novel immune systems targets early infection of nucleus-forming jumbo phages

Jumbo bacteriophages of the [ovbar]KZ-like family are characterized by large genomes (>200 kb) and the remarkable ability to assemble a proteinaceous nucleus-like structure. The nucleus protects the phage genome from canonical DNA-targeting immune systems, such as CRISPR-Cas and restriction-modification. We hypothesized that the failure of common bacterial defenses creates selective pressure for immune systems that target the unique jumbo phage biology. Here, we identify the "jumbo phage killer" (Juk) immune system that is deployed by a clinical isolate of Pseudomonas aeruginosa to resist [ovbar]KZ. Juk immunity rescues the cell by preventing early phage transcription, DNA replication, and nucleus assembly. Phage infection is first sensed by JukA (formerly YaaW), which localizes rapidly to the site of phage infection at the cell pole, triggered by ejected phage factors. The effector protein JukB is recruited by JukA, which is required to enable immunity and the subsequent degradation of the phage DNA. JukA homologs are found in several bacterial phyla and are associated with numerous other putative effectors, many of which provided specific anti-[ovbar]KZ activity when expressed in P. aeruginosa. Together, these data reveal a novel strategy for immunity whereby immune factors are recruited to the site of phage protein and DNA ejection to prevent phage progression and save the cell.

microbiology↗

bioRxiv2022

Hsp90 provides a platform for kinase dephosphorylation by PP5

The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone to maintain kinase proteostasis through the folding and activation of its many client kinases. As with many kinases, the Hsp90 client kinase CRaf is activated by phosphorylation at specific regulatory sites. The cochaperone phosphatase PP5 dephosphorylates CRaf but also Cdc37 in an Hsp90-dependent manner. Although dephosphorylating Cdc37 has been proposed as a mechanism for releasing Hsp90-bound kinases, here we show that Hsp90 bound kinases sterically inhibit Cdc37 dephosphorylation indicating kinase release must occur before Cdc37 dephosphorylation. The cryo-EM structure of PP5 in complex with Hsp90:Cdc37:CRaf reveals how Hsp90 both activates PP5 and scaffolds its association with the bound CRaf to dephosphorylate a site at the C-terminus of the kinase domain. Thus, we directly show how Hsp90s role in maintaining protein homeostasis goes beyond folding and activation to include post translationally modifying its client kinases.

biophysics↗

bioRxiv2022

In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm axonemes, providing the highest resolution structural information to date. Our subtomogram averages reveal mammalian sperm- specific protein complexes within the outer microtubule doublets, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm- specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

biophysics↗

bioRxiv2022

The {varphi}PA3 Phage Nucleus is Enclosed by a Self-Assembling, 2D Crystalline Lattice

A growing number of jumbo bacteriophages, with genomes exceeding 200 kb, have been found to establish a Phage Nucleus--a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host proteins associated with replication and transcription are concentrated inside the Phage Nucleus while nucleotide synthesis, translation, and numerous other host and exogenous proteins are effectively excluded, including CRISPR-Cas and restriction endonuclease host defense systems. Here, we show that fragments of the Phage Nucleus isolated from {phi}PA3 infected Pseudomonas aeruginosa cells form a square lattice and demonstrate that the recombinantly purified primary Phage Nuclear Enclosure (PhuN) protein spontaneously assembles into sheets also constructed from a square lattice which we resolve to 3.8 [A] by cryo-EM. Our structure reveals that the flexible termini and large loops mediate adaptable inter-tetramer contacts that drive shell assembly into a C2-symmetric lattice. While the interfaces between subunits are mostly well packed, two of the interfaces are open, forming clear channels that likely have important functional implications for the transport of proteins, mRNA, and small molecules.

biochemistry↗

bioRxiv2021

Structures of the active HER2/HER3 receptor complex reveal dynamics at the dimerization interface induced by binding of a single ligand

The Human Epidermal Growth Factor Receptor 2 (HER2) and HER3 form a potent pro-oncogenic heterocomplex upon binding of growth factor neuregulin-1{beta} (NRG1{beta})1-3. The mechanism by which HER2 and HER3 interact remains unknown in the absence of any structures of the complex. We isolated the NRG1{beta}-bound near full-length HER2/HER3 dimer and obtained a 2.9[A] cryo-electron microscopy (cryo-EM) reconstruction of the extracellular domain module which reveals unexpected dynamics at the HER2/HER3 dimerization interface. We show that the dimerization arm of NRG1{beta}-bound HER3 is unresolved likely because the apo HER2 monomer fails to undergo a ligand-induced conformational change needed to establish a HER3 dimerization arm binding pocket. In a second structure of an oncogenic extracellular domain mutant of HER2, S310F, we observe a compensatory interaction with the HER3 dimerization arm that stabilizes the dimerization interface. We show that both HER2/HER3 and HER2-S310F/HER3 retain the capacity to bind to the HER2-directed therapeutic antibody, trastuzumab, but the mutant complex does not bind to pertuzumab. Our 3.5[A] structure of the HER2-S310F/HER3/NRG1{beta}/trastuzumab Fragment antigen binding (Fab) complex shows that the receptor dimer undergoes a conformational change to accommodate trastuzumab. Thus, like oncogenic mutations, therapeutics exploit the intrinsic dynamics of the HER2/HER3 heterodimer. The unique features of a singly liganded HER2/HER3 heterodimer underscore the allosteric sensing of the ligand occupancy by the dimerization interface and explain why extracellular domains of HER2 do not homo-associate via canonical active dimer interface.

biophysics↗