Biology subjects

Davis, J. H.

Publications and source records attributed to Davis, J. H..

At least 19 recordsLinked to original sources

bioRxiv2026

Heterogeneous reconstruction algorithms for cryoEM achieve limited particle classification accuracy on real benchmark datasets

The emergence of single-particle cryoEM as a powerful method for structure determination has in large part been fueled by its ability to resolve both single static structures and complex conformational landscapes. Indeed, modern approaches to the heterogeneous reconstruction task can resolve 100s-1,000s of different maps from a single cryoEM dataset. How accurate these algorithms are, however, has proven difficult to rigorously assess, due to a lack of suitable benchmark datasets containing both realistic noise features and ground-truth labels. To address this obstacle, we recently developed a series of benchmark datasets that leverage the targeting power of Cas9 and the programmable heterogeneity of DNA to newly offer access to ground-truth per-particle structural labels in real data. Here, we challenged two popular heterogeneous reconstruction algorithms with mixed particle stacks resampled in silico from these datasets, finding that existing approaches resolve the encoded heterogeneity with limited accuracy. In particular, in realistic particle stacks with complex, multi-scale, and multi-axis heterogeneity, we observed that reconstruction of encoded heterogeneity depended strongly on the application of prior information about where heterogeneity was expected, and that individual particle assignments were made with significant error even when the correct structural states were reconstructed. Both molecular breathing motions and data collection features, such as defocus and projection angle, contributed to the observed particle assignment error. These results highlight important shortcomings of existing heterogeneous reconstruction methods and suggest new avenues for method development in both data collection strategies and in heterogeneous classification and reconstruction algorithms.

biochemistry↗

bioRxiv2026

Engineered Cas9 complexes establish an experimentally grounded benchmark for heterogeneous cryoEM reconstruction methods

Single-particle cryoEM is increasingly used to resolve conformational and compositional ensembles, yet objective evaluation of heterogeneous reconstruction methods remains limited by the scarcity of experimental benchmarks with per-particle ground-truth labels. Indeed, many widely used experimental"benchmark" datasets necessarily validate observed states retrospectively while purely synthetic datasets provide ground-truth labels but typically fail to capture experimentally realistic complexities including confounding structural heterogeneity, imaging noise, contaminants, and orientation biases, which dominate real-world analyses. Here we develop an experimentally grounded benchmark dataset for heterogeneous reconstruction using catalytically inactive Streptococcus pyogenes Cas9 bound to a constant sgRNA and to target DNA duplexes engineered to carry extensions of defined length. We assembled, purified, vitrified, and imaged thirteen complexes independently, such that the dataset-of-origin provides an unambiguous label for each particles encoded state while preserving the full experimental complexity of cryoEM data. Independent refinements of the pure datasets recover the engineered DNA-extension signal and define a simple quantitative readout, DNA-extension occupancy, that increases monotonically with designed extension length. The same reconstructions also reveal substantial non-encoded conformational variability within the Cas9 core, showing that this benchmark embeds a known structural signal within broader structural heterogeneity that methods must confront in practice. To separate these axes of variation, we used systematic deep classification to generate curated particle subsets depleted of selected domain motions while retaining the encoded labels. We further provide pooled particle stacks with standardized per-particle poses in a common reference frame and a lightweight framework for in silico particle pooling to generate challenge datasets with user-defined ground-truth distributions of encoded and non-encoded structural heterogeneity. Together, this resource supports robust benchmarking of heterogeneous reconstruction algorithms and provides a biochemically tractable model system for evaluating entire cryoEM pipelines, including alternative data-collection and preprocessing approaches, under experimentally realistic conditions.

biochemistry↗

bioRxiv2026

A conserved archaeal ribosome-associated factor linking bacterial hibernation and eukaryotic energy sensing

Ribosome hibernation helps cells survive stress by reversibly silencing translation and limiting degradation of ribosomal complexes. Although well characterized in bacteria and eukaryotes, archaeal hibernation remains poorly understood. Using cryoEM to analyze lysates from a model archaeon, we identified AHA (AMPK{gamma}-HPF from Archaea), a broadly conserved ribosome-associated protein factor composed of two distinct modules. Structural analyses showed that AHAs C-terminal domain binds the small subunit, while its N-terminal region recognizes the large subunit, occluding the mRNA channel and the A- and P-tRNA binding sites and thereby enforcing translational silencing. Consistent with this proposed function, {Delta}AHA cells displayed reduced viability, depletion of ribosomal proteins during stationary phase, and impaired recovery upon return to growth. Phylogenetic analyses revealed that AHAs C-terminal domain shares homology with the bacterial Hibernation Promoting Factor (HPF), indicating an origin in the last universal common ancestor (LUCA) and thereby identifying HPF as a universal hibernation module. Strikingly, we observed two AMP molecues bound to AHAs N-terminal CBS-tetrad, which we found was structurally and evolutionary related to the eukaryotic energy sensor AMPK{gamma}, thus linking energy sensing between archaea and eukaryotes. Together, these findings uncover a widespread archaeal ribosome hibernation factor and establish a direct evolutionary link between prokaryotic translational silencing and eukaryotic energy sensing.

biochemistry↗

bioRxiv2026

Ribosome-associated quality control of aberrant protein production during amino acid limitation

Amino acids can become limiting for protein synthesis through depletion of charged tRNAs, leading to ribosome stalling and disruption of translation elongation at specific codons. To assess whether this is a mechanism by which amino acid availability can directly influence gene expression, we designed a reporter library to measure translation disruption across all sense codons in the context of amino acid limitations. We found that arginine limitation consistently impairs translation at the arginine codon AGA, resulting in synthesis of proteins from endogenous transcripts. In contrast, GCN2 pathway activation suppresses translation disruption following depletion of most other amino acids. Genome-wide screens revealed that the ribosome quality control trigger (RQC-T) and RQC pathways, which resolve ribosome collisions on defective mRNAs, catalyze ribosome splitting and premature fall-off in response to arginine depletion. Additionally, the E3 ubiquitin ligase RNF14, recently shown to clear ribosome A-site obstructions, promotes translation disruption through both ribosome fall-off and frameshifting during arginine limitation. Together, these data show that the RQC machinery is engaged by tRNA-limited ribosomes and identify a new role for RNF14 as a regulator of translation upon arginine limitation.

genetics↗

bioRxiv2026

A proteome-wide biochemical screen defines binding determinants of the core autophagy protein LC3B

Human MAP1LC3B (LC3B) binds proteins involved in autophagy and other cellular processes using a degenerate four-residue short linear motif known as the LC3-interacting region (LIR). Biochemical and structural studies have identified LIRs in many LC3B interaction partners, but the sequence features that contribute to binding have not been systematically explored. To discover peptides that interact with LC3B and deeply profile the key binding determinants, we screened a library of ~500,000 36-residue peptides derived from the human proteome using bacterial cell-surface display. Analysis of the screening data, coupled with structural studies and site-directed mutagenesis, revealed exceptions to the canonical LIR motif ([FWY]0-X1-X2-[LVI]3) and a strong preference for negatively charged residues adjacent to the LIR, which we visualized in a newly determined structure of LC3B bound to a peptide isolated in our screen. Guided by our screening data, we designed synthetic LIR-containing peptides that bind LC3B with affinities comparable to the tightest measured natural binder. Finally, we determined that mutations in LC3B commonly thought to abrogate binding of LIR-containing peptides instead alter LC3B binding specificity, leading to enhanced binding of some LIR-containing sequences. Taken together, our results refine the LIR motif definition, expand the network of candidate LC3B interaction partners, and highlight how mutations at the LIR docking site can modulate affinity and specificity.

biochemistry↗

bioRxiv2025

Capturing ribosomal structures in cellular extracts with cryoPRISM: A purification-free cryoEM approach reveals novel structural states

Structural analyses of ribosomes by single particle cryogenic electron microscopy (cryoEM) have traditionally relied on purified or reconstituted samples, with particles often trapped in desired states using genetic, pharmacological, or biochemical perturbations. While informative, such in vitro methods often fail to capture the full diversity of structural states and associated protein factors present in cells. In contrast, in situ cryoelectron tomography preserves cellular context but is limited by low throughput and modest resolution. Here, we present cryoPRISM (purification-free ribosome imaging from subcellular mixtures), a rapid ex vivo workflow encompassing cell lysis, vitrification, and image analysis methods for high-resolution analyses of ribosomal structures directly from cell lysates. Applying cryoPRISM in E. coli, we resolved more than twenty distinct ribosomal states spanning assembly, translation initiation, elongation, trans-translation, and quiescence, including a novel configuration of EF-G bound to idle ribosomes with the ribosome hibernation factor RaiA. Given its speed, accessibility, and ability to preserve native interactions and structural heterogeneity, we anticipate that cryoPRISM will be broadly applicable for uncovering ribosomal biology across diverse organisms and conditions.

biochemistry↗

bioRxiv2024

Automated model-free analysis of cryo-EM volume ensembles with SIREn

Cryogenic electron microscopy (cryo-EM) has the potential to capture snapshots of proteins in motion and generate hypotheses linking conformational states to biological function. This potential has been increasingly realized by the advent of machine learning models that allow 100s-1,000s of 3D density maps to be generated from a single dataset. How to identify distinct structural states within these volume ensembles and quantify their relative occupancies remain open questions. Here, we present an approach to inferring variable regions directly from a volume ensemble based on the statistical co-occupancy of voxels, as well as a 3D-convolutional neural network that predicts binarization thresholds for volumes in an unbiased and automated manner. We show that these tools recapitulate known heterogeneity in a variety of simulated and real cryo-EM datasets, and highlight how integrating these tools with existing data processing pipelines enables improved particle curation and the construction of quantitative conformational landscapes.

biochemistry↗

bioRxiv2024

Bulk and selective autophagy cooperate to remodel a fungal proteome in response to changing nutrient availability

Cells remodel their proteomes in response to changing environments by coordinating changes in protein synthesis and degradation. In yeast, such degradation involves both proteasomal and vacuolar activity, with a mixture of bulk and selective autophagy delivering many of the vacuolar substrates. Although these pathways are known to be generally important for such remodeling, their relative contributions have not been reported on a proteome-wide basis. To assess this, we developed a method to pulse-label the methylotrophic yeast Komagataella phaffii (i.e. Pichia pastoris) with isotopically labeled nutrients, which, when coupled to quantitative proteomics, allowed us to globally monitor protein degradation on a protein-by-protein basis following an environmental perturbation. Using genetic ablations, we found that a targeted combination of bulk and selective autophagy drove the vast majority of the observed proteome remodeling activity, with minimal non-autophagic contributions. Cytosolic proteins and protein complexes, including ribosomes, were degraded via Atg11-independent bulk autophagy, whereas proteins targeted to the peroxisome and mitochondria were primarily degraded in an Atg11-dependent manner. Notably, these degradative pathways were independently regulated by environmental cues. Taken together, our new approach greatly increases the range of known autophagic substrates and highlights the outsized impact of autophagy on proteome remodeling. Moreover, the resulting datasets, which we have packaged in an accessible online database, constitute a rich resource for identifying proteins and pathways involved in fungal proteome remodeling.

bioengineering↗

bioRxiv2024

An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins

FtsH, a AAA protease, associates with HflK/C subunits to form a megadalton complex that spans the inner membrane and extends into the periplasm of E. coli. How this complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following overproduction of protein components, recent cryo-EM structures reveal symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit degradation of membrane-embedded substrates. Here, we present structures of native complexes in which HflK/C instead forms an asymmetric nautilus-like assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. The membrane curvature in our FtsH{middle dot}HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid-scramblase activity and possibly with FtsHs function in the degradation of membrane-embedded proteins.

biochemistry↗

bioRxiv2024

Integrated proteasomal and lysosomal activity shape mTOR-regulated proteome remodeling

The crucial growth regulator mTOR is suppressed during nutrient limitation, which reduces protein synthesis and activates the ubiquitin-proteasome system (UPS) and lysosomal degradation pathways. Whereas these pathways have been extensively studied individually, their integrated dynamics, including the interplay between protein synthesis and degradation, and the coordination between lysosomal and UPS pathways, remain underexplored. Here, we couple stable isotope pulse-labeling and mass spectrometry to quantify and kinetically model proteome dynamics following mTOR inhibition in cultured human cells. Using a combination of genetics and pharmacological inhibitors, we identify proteins strictly degraded by one pathway, those that undergo multimodal degradation, and others that can flexibly access the proteasome or lysosome subject to the availability of either. Our data resource, comprised of [~]5.2 million proteomic measurements, reveals that the UPS and lysosomal pathway operate with disparate kinetics, and highlights the rapid nature of lysosomal degradation. Additionally, we observe that cells coordinate the synthesis and degradation of translation initiation and elongation factors, leading to preferential synthesis from key classes of mRNA transcripts. Taken together, this work illuminates the complex, integrated pathways influencing proteostasis when mTOR is inhibited, provides a rich resource detailing the kinetics of protein synthesis and degradation, and establishes a robust methodology for measuring proteome dynamics on a per-protein basis in the context of cellular stress.

biochemistry↗

bioRxiv2024

NCOA4 initiates ferritinophagy by avidly binding GATE16 using two short linear interaction motifs

Cells carefully regulate cytosolic iron, which is a vital enzymatic cofactor, yet is toxic in excess. In mammalian cells, surplus iron is sequestered in ferritin cages that, in iron limiting conditions, are degraded through the selective autophagy pathway ferritinophagy to liberate free iron. Prior work identified the ferritinophagy receptor protein NCOA4, which links ferritin and LC3/GABARAP-family member GATE16, effectively tethering ferritin to the autophagic machinery. Here, we elucidate the molecular mechanism underlying this interaction, discovering two short linear motifs in NCOA4 that each bind GATE16 with weak affinity. These binding motifs are highly avid and, in concert, support high-affinity NCOA4*GATE16 complex formation. We further find the minimal NCOA4383-522 fragment bearing these motifs is sufficient for ferritinophagy and that both motifs are necessary for this activity. This work suggests a general mechanism wherein selective autophagy receptors can distinguish between the inactive soluble pools of LC3/GABARAPs and the active membrane-conjugated forms that drive autophagy. Finally, we find that iron decreases NCOA4383-522s affinity for GATE16, providing a plausible mechanism for iron-dependent regulation of ferritinophagy.

biochemistry↗

bioRxiv2024

Bispecific GD2 x B7-H3 Antibody Improves Tumor Targeting and Reduces Toxicity while Maintaining Efficacy for Neuroblastoma

The current treatment for neuroblastoma involves an immunotherapy regimen that includes a monoclonal antibody that recognizes disialoganglioside (GD2), expressed at high levels on neuroblastoma. GD2 is not present on most normal tissues but is expressed on nerves. Thus, anti-GD2 treatment causes substantial, dose-limiting, neuropathic pain. B7-H3 is overexpressed on multiple tumor types, including neuroblastoma, with minimal normal cell expression and is absent on nerves. We designed a bispecific antibody (bsAb) that requires simultaneous binding of these two tumor antigens to achieve tight-binding of tumor cells. Our preclinical research shows that when compared to an anti-GD2 monospecific antibody, the GD2xB7-H3 bsAb has improved tumor specificity with similar efficacy and reduced toxicity. Since this bsAb does not bind to nerves, it may be possible to administer increased or additional doses beyond the tolerable dose of monospecific anti-GD2 antibodies, which could improve therapeutic efficacy and quality of life for patients with neuroblastoma.

cancer biology↗

bioRxiv2024

Rapid structural analysis of bacterial ribosomes in situ

Rapid structural analysis of purified proteins and their complexes has become increasingly common thanks to key methodological advances in cryo-electron microscopy (cryo-EM) and associated data processing software packages. In contrast, analogous structural analysis in cells via cryo-electron tomography (cryo-ET) remains challenging due to critical technical bottlenecks, including low-throughput sample preparation and imaging, and laborious data processing methods. Here, we describe the development of a rapid in situ cryo-ET sample preparation and data analysis workflow that results in the routine determination of sub-nm resolution ribosomal structures. We apply this workflow to E. coli, producing a 5.8 [A] structure of the 70S ribosome from cells in less than 10 days, and we expect this workflow will be widely applicable to related bacterial samples.

cell biology↗

bioRxiv2023

A proteolytic AAA+ machine poised to unfold a protein substrate

AAA+ proteolytic machines unfold proteins prior to degradation. Cryo-EM of a ClpXP-substrate complex reveals a postulated but heretofore unseen intermediate in substrate unfolding/degradation. The natively folded substrate is drawn tightly against the ClpX channel by interactions between axial pore loops and the substrate degron tail, and by contacts with the native substrate that are, in part, enabled by movement of one ClpX subunit out of the typically observed hexameric spiral.

molecular biology↗

bioRxiv2023

Application of monolayer graphene to cryo-electron microscopy grids for high-resolution structure determination

In cryogenic electron microscopy (cryo-EM), purified macromolecules are typically applied to a grid bearing a holey carbon foil, blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice that is suspended across roughly 1 m-wide foil holes. The resulting sample is then imaged using cryogenic transmission electron microscopy and, after substantial image processing, near-atomic resolution structures can be determined. Despite cryo-EMs widespread adoption, sample preparation remains a severe bottleneck in cryo-EM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryo-EM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryo-EM grids, and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 [A] resolution density map of an exemplar Cas9 complex using a highly pure sample at a relatively low concentration. SUMMARYThe application of support layers, such as graphene, to cryo-electron microscopy grids can increase the density of particles imaged, limit particle interactions with the air-water interface, reduce the extent of beam-induced motion, and, in some instances, improve the distribution of particle orientations. This paper describes a robust protocol for coating cryo-EM grids with a monolayer of graphene for improved cryo-sample preparation.

biochemistry↗

bioRxiv2023

Learning structural heterogeneity from cryo-electron sub-tomograms with tomoDRGN

Cryo-electron tomography (cryo-ET) allows one to observe macromolecular complexes in their native, spatially contextualized environment. Tools to visualize such complexes at nanometer resolution via iterative alignment and averaging are well-developed but rely on assumptions of structural homogeneity among the complexes under consideration. Recently developed downstream analysis tools allow for some assessment of macromolecular diversity but have limited capacity to represent highly heterogeneous macromolecules, including those undergoing continuous conformational changes. Here, we extend the highly expressive cryoDRGN deep learning architecture, originally created for cryo-electron microscopy single particle analysis, to sub-tomograms. Our new tool, tomoDRGN, learns a continuous low-dimensional representation of structural heterogeneity in cryo-ET datasets while also learning to reconstruct a large, heterogeneous ensemble of structures supported by the underlying data. Using simulated and experimental data, we describe and benchmark architectural choices within tomoDRGN that are uniquely necessitated and enabled by cryo-ET data. We additionally illustrate tomoDRGNs efficacy in analyzing an exemplar dataset, using it to reveal extensive structural heterogeneity among ribosomes imaged in situ.

molecular biology↗

bioRxiv2022

The SspB adaptor drives structural changes in the AAA+ ClpXP protease during ssrA-tagged substrate delivery

Energy-dependent protein degradation by the AAA+ ClpXP protease helps maintain protein homeostasis in organisms ranging from simple bacteria to humans. In E. coli and many other proteobacteria, the SspB adaptor assists ClpXP in degrading ssrA-tagged polypeptides produced as a consequence of tmRNA-mediated ribosome rescue. By tethering these incomplete ssrA-tagged proteins to ClpXP, SspB facilitates their efficient degradation at low substrate concentrations. How this process occurs structurally is unknown. Here, we present a cryo-EM structure of the SspB adaptor bound to a GFP-ssrA substrate and to ClpXP. This structure provides evidence for simultaneous contacts of SspB and ClpX with the ssrA tag within the tethering complex, allowing direct substrate handoff concomitant with the initiation of substrate translocation. Furthermore, our structures reveal that binding of the substrate*adaptor complex induces unexpected conformational changes within the spiral structure of the AAA+ ClpX hexamer and its interaction with the ClpP tetradecamer. SIGNIFICANCEIntercellular proteases, including ClpXP, degrade damaged or unneeded proteins. Peptide tags allow specific protein substrates to be recognized by the ClpX unfoldase/translocase component of ClpXP and by an adaptor, SspB, which tethers itself to ClpX and enhances ClpXP degradation of the tagged protein. Our cryo-EM structure of ClpXP bound to SspB and a tagged substrate shows that SspB and ClpX simultaneously contact the degradation tag and reveal changes in the structure of ClpX and its interaction with ClpP. These structural changes appear to be a prelude to an initial ClpX translocation step that pulls the substrate away from SspB and initiates degradation by allowing substrate unfolding and further translocation of the unfolded substrate into the proteolytic chamber of ClpP.

biochemistry↗

bioRxiv2022

A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation

Intracellular proteases must be specific to avoid degrading the wrong proteins. Here, we present cryo-EM structures of E. coli ClpXP, a AAA+ protease, which reveal that the axial channel of ClpX is closed prior to the binding and subsequent translocation of a protein substrate. An open-channel ClpX mutation stimulates degradation of casein, a non-specific substrate, indicating that channel closure contributes to increased degradation specificity. We demonstrate that ClpX activates ClpP cleavage of a degron-free decapeptide by a channel-independent mechanism, in which the peptide substrate appears to pass through a symmetry mismatched gap in the interface between ClpX and ClpP before entering the degradation chamber via the axial portal of ClpP. The peptide products of ClpXP protein degradation are likely to exit the chamber by the reverse route.

biochemistry↗