Biology subjects

Agard, D.

Publications and source records attributed to Agard, D..

At least 19 recordsLinked to original sources

bioRxiv2026

A tripartite mechanism licenses RNA polymerase import into the phage nucleus

Nucleus-forming jumbo phages import a phage-encoded non-virion RNA polymerase (nvRNAP) into a proteinaceous nucleus, but how it is selected for import is unknown. Here, we identify an import pathway that couples nvRNAP subunit interactions with phage-encoded import factors. Maximal nvRNAP import requires a novel factor Imp7 (gp166) and the essential import factor Imp1. Imp7 is essential at environmental temperatures (20 C), where impaired nvRNAP import causes a profound loss of middle and late transcription and blocks phage DNA replication. Import of individual nvRNAP subunits also depends on other subunits, consistent with import licensing at the level of an assembled complex. In the absence of middle transcription and DNA replication, the phage nucleus surprisingly still assembles and segregates from the cytoplasm, demonstrating that nvRNAP nuclear localization and activity are not a critical checkpoint for nucleus assembly. Together, these findings define a specialized pathway for nuclear import of the multi-subunit RNAP and reveal that nuclear compartment assembly precedes the transcriptional and replicative programs required for its maturation.

microbiology↗

bioRxiv2026

A Crossed Laser Phase Plate for CryoEM

The laser phase plate (LPP) enables phase-contrast imaging in cryogenic electron microscopy (cryoEM), enhancing image contrast without compromising high-resolution information. Here we report the implementation of a crossed laser phase plate (xLPP) comprising two optical cavities oriented orthogonally, installed in a ThermoFisher Scientific Krios G4 microscope equipped with a newly designed transfer lens module. We demonstrate the expected, strong contrast enhancement and stable, additive phase shifts of 90{degrees}, with a contrast transfer function (CTF) that closely matches theory. Single-particle analysis (SPA) of apoferritin, a standard benchmark sample, reached a resolution of 1.79 [A], demonstrating the system is capable of acquiring high-resolution cryoEM data. When imaging thick E. coli cells ([~]350 nm), the xLPP enhances contrast and increases low-frequency template-matching signal. Together, these results establish the feasibility of the xLPP and highlight its potential for high-contrast, high-resolution cryoEM imaging of biological systems.

biophysics↗

bioRxiv2026

Characterization Standard for In-situ Cryo-electron Tomography

Standardized biological specimens are essential for optimizing cryoEM workflows and benchmarking instrument performance. While apoferritin fulfills this role for single-particle analysis, no equivalent exists for cryo-electron tomography. Ribosomes are frequently used but require large datasets due to C1 symmetry and structural heterogeneity, limiting rapid optimization and standardized comparison of workflows. Here, we present PP7 virus-like particles (VLPs) overexpressed in E. coli as a scalable in situ benchmark. VLPs have high orders of symmetry enabling rapid, high-resolution validation of tomographic pipelines from minimal datasets, while their distinct structural features across low to high resolutions provide a practical resolution metric.

cell biology↗

bioRxiv2024

Annotating CryoET Volumes: A Machine Learning Challenge

Cryo-electron tomography (cryoET) has emerged as a powerful structural biology tool for understanding protein complexes in their native cellular environments. Presently, 3D volumes of cellular environments can be acquired in the thousands in a few days where each volume provides a rich and complex cellular landscape. Despite numerous innovations, localizing and identifying the vast majority of protein species in these volumes remains prohibitively difficult. Machine learning based methods provide an opportunity to automate the process of labeling and annotating cryoET volumes. Due to current bottlenecks in the annotation process, and a lack of large standardized datasets, training datasets for machine learning algorithms have been scarce. Here, we present a defined "phantom" sample, along with "ground truth" annotations, that will be the basis of a machine learning challenge to bring cryoET and ML experts together and spur creativity to address this annotation problem. We have also set up a cryoET data portal that provides additional diverse sets of annotated 3D volumes from cryoET experts across the world for the machine learning challenge.

cell biology↗

bioRxiv2024

Real-space heterogeneous reconstruction, refinement, and disentanglement of CryoEM conformational states with HetSIREN

Single-particle analysis by Cryo-electron microscopy (CryoEM) provides direct access to the conformation of each macromolecule. However, the images signal-to-noise ratio is low, and some form of classification is usually performed at the image processing level to allow structural modeling. Classical classification methods imply the existence of a discrete number of structural conformations. However, new heterogeneity algorithms introduce a novel reconstruction paradigm, where every state is represented by a lower number of particles, potentially just one, allowing the estimation of conformational landscapes representing the different structural states a biomolecule explores. In this work, we present a novel deep learning-based method called HetSIREN. HetSIREN can fully reconstruct or refine a CryoEM volume in real space based on the structural information summarized in a conformational latent space. The unique characteristics that set HetSIREN apart start with the definition of the approach as a real space-based only method, a fact that allows spatially focused analysis, but also the introduction of a novel network architecture specifically designed to make use of meta-sinusoidal activations, with proven high analytics capacities. Continuing with innovations, HetSIREN can also refine the pose parameters of the images at the same time that it conditions the network with prior information/constraints on the maps, such as Total Variation and L1 denoising, ultimately yielding cleaner volumes with high-quality structural features. Finally, but very importantly, HetSIREN addresses one of the most confusing issues in heterogeneity analysis, as it is the fact that real structural heterogeneity estimation is entangled with pose estimation (and to a lesser extent with CTF estimation), in this way, HetSIREN introduces a novel encoding architecture able to decouple pose and CTF information from the conformational landscape, resulting in more accurate and interpretable conformational latent spaces. We present results on computer-simulated data, public data from EMPIAR, and data from experimental systems currently being studied in our laboratories. An important finding is the sensitivity of the structure and dynamics of the SARS-CoV-2 Spike protein on the storage temperature.

bioinformatics↗

bioRxiv2024

Defining the constituents and functions of a lipid-based jumbo phage compartment

Viruses are vulnerable to cellular defenses at the start of the infection when a single copy genome has not yet initiated de novo viral protein synthesis. A family of jumbo phages related to phage {Phi}KZ, which infects Pseudomonas aeruginosa, uses a protein-based phage nucleus to protect the DNA during middle and late stages of infection, but how it is protected prior to phage nucleus assembly is unclear. To reveal the environment surrounding injected phage DNA, we determine which proteins interact with phage proteins injected with the phage genome. Here, we surprisingly identify host proteins related to membrane and lipid biology co-purifying with the injected phage protein. Staining of infected cells with lipophilic dyes revealed a clustering of host lipids co-localized with phage DNA and protein in an early phage infection (EPI) vesicle. Early gene expression is induced by the co-injected virion RNAP (vRNAP), with specific early proteins then associating with the EPI vesicle and ribosomes, likely to facilitate localized translation. As the infection progresses, the phage genome separates from the EPI vesicle and vRNAP, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases notably do not localize to the EPI vesicle, supporting that DNA is not exposed at this early time point. We therefore propose that the EPI vesicle is rapidly constructed together with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure the faithful transfer of the single copy phage genome to the proteinaceous nucleus.

microbiology↗

bioRxiv2023

Poc1 is a basal body inner junction protein that promotes triplet microtubule integrity and interconnections

Basal bodies (BBs) are conserved eukaryotic structures that organize motile and primary cilia. The BB is comprised of nine, cylindrically arranged, triplet microtubules (TMTs) that are connected to each other by inter-TMT linkages which maintain BB structure. During ciliary beating, forces transmitted to the BB must be resisted to prevent BB disassembly. Poc1 is a conserved BB protein important for BBs to resist ciliary forces. To understand how Poc1 confers BB stability, we identified the precise position of Poc1 binding in the Tetrahymena BB and the effect of Poc1 loss on BB structure. Poc1 binds at the TMT inner junctions, stabilizing TMTs directly. From this location, Poc1 also stabilizes inter-TMT linkages throughout the BB, including the cartwheel pinhead and the inner scaffold. Moreover, we identify a molecular response to ciliary forces via a molecular remodeling of the inner scaffold, as determined by differences in Fam161A localization. Thus, while not essential for BB assembly, Poc1 promotes BB interconnections that establish an architecture competent to resist ciliary forces.

cell biology↗

bioRxiv2023

Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping

Host-pathogen interactions (HPIs) are pivotal in regulating establishment, progression, and outcome of an infection. Affinity-purification mass spectrometry has become instrumental for the characterization of HPIs, however the targeted nature of exogenously expressing individual viral proteins has limited its utility to the analysis of relatively small pathogens. Here we present the use of co-fractionation mass spectrometry (SEC-MS) for the high-throughput analysis of HPIs from native viral infections of two jumbophages ({phi}KZ and{phi} PA3) in Pseudomonas aeruginosa. This enabled the detection >6000 unique host-pathogen and >200 pathogen-pathogen interactions for each phage, encompassing >50% of the phage proteome. Interactome-wide comparison across phages showed similar perturbed protein interactions suggesting fundamentally conserved mechanisms of phage predation within the KZ-like phage family. Prediction of novel ORFs revealed a{phi} PA3 complex showing strong structural and sequence similarity to{phi} KZ nvRNAp, suggesting{phi} PA3 also possesses two RNA polymerases acting at different stages of the infection cycle. We further expanded our understanding on the molecular organization of the virion packaged and injected proteome by identifying 23 novel virion components and 5 novel injected proteins, as well as providing the first evidence for interactions between KZ-like phage proteins and the host ribosome. To enable accessibility to this data, we developed PhageMAP, an online resource for network query, visualization, and interaction prediction (https://phagemap.ucsf.edu/). We anticipate this study will lay the foundation for the application of co-fractionation mass spectrometry for the scalable profiling of hostpathogen interactomes and protein complex dynamics upon infection.

systems biology↗

bioRxiv2022

AreTomo: An integrated software package for automated marker-free, motion-corrected cryo-electron tomographic alignment and reconstruction

AreTomo, an abbreviation for Alignment and Reconstruction for Electron Tomography, is a GPU accelerated software package that fully automates motion-corrected marker-free tomographic alignment and reconstruction in a single package. By correcting in-plane rotation, translation, and importantly, the local motion resulting from beam-induced motion from tilt to tilt, AreTomo can produce tomograms with sufficient accuracy to be directly used for subtomogram averaging. Another major application is the on-the-fly reconstruction of tomograms in parallel with tilt series collection to provide users with real-time feedback of sample quality allowing users to make any necessary adjustments of collection parameters. Here, the multiple alignment algorithms implemented in AreTomo are described and the local motions measured on a typical tilt series are analyzed. The residual local motion after correction for global motion was found in the range of {+/-}80 [A], indicating that the accurate correction of local motion is critical for high-resolution cryo-electron tomography (cryoET).

cell biology↗

bioRxiv2022

Structural features of mTORC2 that control substrate-specific activities.

mTORC2 is a multi-subunit kinase complex that is central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1 distinguish it from its relative, mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously, however, important questions remain, particularly regarding structural determinants of substrate specificity and context dependent activities. We used cryo-EM to obtain high resolution structures of the human mTORC2 apo-complex, as well as structures in the presence of substrates, Akt and SGK1. Specific predictions suggested by substrate-induced structural changes were tested in functional assays. First, side chain interactions between Rictor and mTOR that prevent recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin were visualized for the first time in the apo-state, demonstrating the steric occlusion that prevents mTORC2 interaction with mTORC1 substrates and rapamycin. Also in the apo-state, mSin1 was seen to form extensive contacts with Rictor, including a pair of short -helices nestled between two Rictor helical repeat clusters, followed by an extended strand, which makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1/Arg-83, which comes to interact with a negative patch within Rictor. Mutation of Arg-83 to Ala selectively disrupted mTORC2 dependent phosphorylation of SGK1 but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activities.

biochemistry↗

bioRxiv2021

Electron Cryo-Tomography Structure of Axonemal Doublet Microtubule from Tetrahymena thermophila

Doublet microtubules (DMT) provide a scaffold for axoneme assembly in motile cilia. Aside from /{beta} tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMT, collectively named the microtubule inner proteins (MIPs). We used electron cryo-tomography to study axoneme DMT isolated from Tetrahymena thermophila. We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP Rib72A/B binds to the luminal wall of the DMT by multiple DM10 domains, likely by recognizing the acetylated K40 residue of -tubulin. We found Fap115, a MIP containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of the A-tubule. It functions as a "molecular staple" stabilizing both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. Defects caused by the depletion of Fap115 propagate along the axoneme due to extensive structural changes in the DMT at and beyond the Fap115 binding site. Finally, by comparing DMT structures from Tetrahymena and Chlamydomonas, we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.

cell biology↗

bioRxiv2020

Cryo-EM reveals the dynamic interplay between mitochondrial Hsp90 and SdhB folding intermediates

TRAP1 is a mitochondrion specific Hsp90, a ubiquitous chaperone family that mediates the folding and maturation of hundreds of "client" proteins. Through the interaction with client proteins, TRAP1 regulates mitochondrial protein homeostasis, oxidative phosphorylation/glycolysis balance, and plays a critical role in mitochondrial dynamics and disease. However, the molecular mechanism of client protein recognition and remodeling by TRAP1 remains elusive. Here we established the succinate dehydrogenase B subunit (SdhB) from mitochondrial complex II as a client protein for TRAP1 amenable to detailed biochemical and structural investigation. SdhB accelerates the rate of TRAP1 dimer closure and ATP hydrolysis by 5-fold. Cryo-EM structures of the TRAP1:SdhB complex show TRAP1 stabilizes SdhB folding intermediates by trapping an SdhB segment in the TRAP1 lumen. Unexpectedly, client protein binding induces an asymmetric to symmetric transition in the TRAP1 closed state. Our results highlight a client binding mechanism conserved throughout Hsp90s that transcends the need for cochaperones and provide molecular insights into how TRAP1 modulates protein folding within mitochondria. Our structures also suggest a potential role for TRAP1 in Fe-S cluster biogenesis and mitochondrial protein import and will guide small molecule development for therapeutic intervention in specific TRAP1 client interactions.

biophysics↗

bioRxiv2020

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

Motile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia of Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout (KO) Tetrahymena cells. From this analysis we identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

molecular biology↗

bioRxiv2020

Cryo-EM structures reveal a multistep mechanism of Hsp90 activation by co-chaperone Aha1

Hsp90 is a ubiquitous molecular chaperone that facilitates the folding and maturation of hundreds of cellular “client” proteins. The ATP-driven client maturation process is regulated by a large number of co-chaperones. Among them, Aha1 is the most potent activator of Hsp90 ATPase activity and thus dramatically affects Hsp90’s client proteins. To understand the Aha1 activation mechanism, we determined full-length Hsp90:Aha1 structures in six different states by cryo-electron microscopy, including nucleotide-free semi-closed, nucleotide-bound pre-hydrolysis, and semi-hydrolyzed states. Our structures demonstrate that the two Aha1 domains can each interact with Hsp90 in two different modes, uncovering a complex multistep activation mechanism. The results show that Aha1 accelerates the chemical step of ATP hydrolysis like a conventional enzyme, but most unusually, catalyzes the rate-limiting large-scale conformational changes of Hsp90 fundamentally required for ATP hydrolysis. Our work provides a structural framework to guide small molecule development targeting this critical modulator of client protein maturation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

XMAP215 and γ-tubulin additively promote microtubule nucleation in purified solutions

Microtubule nucleation is spatiotemporally regulated in cells by several molecules, including the template {gamma}-tubulin and the polymerase XMAP215. Recently, XMAP215 and the {gamma}-tubulin ring complex were reported to function synergistically, and this synergy was hypothesized to be due to direct binding between XMAP215 and {gamma}-tubulin. Here, we address this hypothesis by 1) probing domain requirements for XMAP215 to promote microtubule nucleation and 2) testing whether XMAP215 functions synergistically with {gamma}-tubulin in the absence of the other ring complex proteins. We confirm that {gamma}-tubulin and XMAP215 are classically defined nucleators that reduce the nucleation lag seen in bulk tubulin assembly. Then, using deletion constructs, we show that XMAP215s ability to nucleate microtubules in purified solutions correlates with its ability to elongate existing microtubules and does not depend on the number of TOG domains. Finally, we show that XMAP215 and {gamma}-tubulin promote {beta}-tubulin assembly in an additive, not synergistic, manner. Thus, their modes of action during microtubule nucleation are distinct, and the synergy reported between XMAP215 and the {gamma}-tubulin ring complex is not due to {gamma}-tubulin alone.

biochemistry↗

bioRxiv2020

The interplay between Bag-1, Hsp70, and Hsp90 reveals that inhibiting Hsp70 rebinding is essential for Glucocorticoid Receptor activity

The glucocorticoid receptor (GR), like many signaling proteins requires Hsp90 for sustained activity. Previous biochemical studies revealed that the requirement for Hsp90 is explained by its ability to reverse Hsp70-mediated inactivation of GR through a complex process requiring both cochaperones and Hsp90 ATP hydrolysis. How ATP hydrolysis on Hsp90 enables GR reactivation is unknown. The canonical mechanism of client release from Hsp70 requires ADP:ATP exchange, which is normally rate limiting. Here we show that independent of ATP hydrolysis, Hsp90 acts as an Hsp70 nucleotide exchange factor (NEF) to accelerate ADP dissociation, likely coordinating GR transfer from Hsp70 to Hsp90. As Bag-1 is a canonical Hsp70 NEF that can also reactivate Hsp70:GR, the impact of these two NEFs was compared. Simple acceleration of Hsp70:GR release was insufficient for GR reactivation as Hsp70 rapidly re-binds and re-inactivates GR. Instead, inhibition of GR re-inactivation by Hsp70 is critical. This can be accomplished by high non-physiological Bag-1 concentrations, which also inhibit Hsp70:ATP binding. In contrast, in an ATP-hydrolysis dependent process, Hsp90 plays a unique role by kinetically partitioning GR into a state that can bind ligand, but is protected from Hsp70 inactivation, thus allowing GR to be activated by its ligand but still able to re-enter the chaperone cycle. At physiologic concentrations, Bag-1 works synergistically with Hsp90 to accelerate the first rate-limiting step in GR reactivation. The net effect is that the chaperone machinery cyclically dictates the on and off rates for GR ligand, providing a timer controlling the persistence of activated GR. Significance StatementThe glucocorticoid receptor (GR) is an essential transcription regulatory factor. Like many signaling proteins, GR activity is regulated by two essential molecular chaperones: Hsp90 and Hsp70. Functioning like a toggle switch, Hsp70 first inactivates GR, and then Hsp90 reactivates GR in an Hsp90 ATP hydrolysis dependent manner. Here, an intricate set of biochemistry experiments uncover fundamental principles governing how these chaperone systems collaboratively regulate GR activity. While Hsp90 promotes GR release from Hsp70 by modulating Hsp70s nucleotide state, this occurs independently of Hsp90 ATP hydrolysis. Instead, ATP hydrolysis on Hsp90 facilitates a second essential reactivation step resulting in an Hsp90-bound GR state that protects GR from Hsp70 re-inactivation. A kinetic partitioning model best describes chaperone modulation of GRs activity.

biochemistry↗

bioRxiv2019

Amino and PEG-Amino Graphene Oxide Grids Enrich and Protect Samples for High-resolution Single Particle Cryo-electron Microscopy

Cryo-EM samples prepared using the traditional methods often suffer from too few particles, poor particle distribution, or strongly biased orientation, or damage from the air-water interface. Here we report that functionalization of graphene oxide (GO) coated grids with amino groups concentrates samples on the grid with improved distribution and orientation. By introducing a PEG spacer, particles are kept away from both the GO surface and the air-water interface, protecting them from potential denaturation.

biophysics↗

bioRxiv2019

General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM

Despite their great potential to facilitate rapid preparation of quite impure samples, affinity grids have not yet been widely employed in single particle cryo-EM. Here, we chemically functionalize graphene oxide coated grids and use a highly specific covalent affinity tag system. Importantly, our polyethylene glycol spacer keeps particles away from the air-water interface and graphene oxide surface, protecting them from denaturation or aggregation and permits high-resolution reconstructions of small particles.

biophysics↗