Biology subjects

Herzik, M. A.

Publications and source records attributed to Herzik, M. A..

17 recordsLinked to original sources

bioRxiv2026

Glk1p is an actin fold metabolic enzyme whose polymerization is sensitive to nucleotide state

The actin fold is present in enzymes ranging from sugar kinases to chaperones. Previous work on the actin fold metabolic enzyme, glucokinase (Glk1p) in S. cerevisiae found it could form filaments in response to its substrates, ATP and glucose (Stoddard et al., 2020). Here, we have identified the product, glucose 6-phosphate (G6P), as a second trigger for Glk1p polymerization in vitro. Furthermore, the addition of ADP to G6P-Glk1p filaments causes filament disassembly, suggesting that polymerization is sensitive to the state of the bound nucleotide and/or the transfer of the gamma phosphate. We have also identified a specific metabolic state, the accumulation of G6P during stationary phase, that triggers Glk1p polymerization in vivo. While the structures of Glk1p filaments in either the ATP/glucose or G6P-bound form are not similar to conventional actin filaments, the sensitivity of assembly to the gamma phosphate of the nucleotide provides a conceptual bridge between the cytoskeleton and metabolic regulation via enzyme polymerization. SummaryThe yeast glucokinase, Glk1p, is an actin fold enzyme that undergoes regulated polymerization in response to ATP/glucose and glucose 6-phosphate (G6P). Elevated G6P during stationary phase triggers Glk1p filament assembly in vivo consistent with a role in regulating glycolytic flux.

cell biology↗

bioRxiv2026

A Surfactant Cocktail Overcomes Air-Water Interface Artifacts in Single-Particle CryoEM

Single-particle cryogenic electron microscopy (cryoEM) is a widely used technique for structure determination of biomacromolecules to near-atomic resolution. Random distributions of these molecules in vitrified ice are necessary to accumulate enough two-dimensional views to generate a complete three-dimensional (3-D) reconstruction. However, interactions between the sample and the air-water interface (AWI) that occur during vitrification often bias the views of the sample, a phenomenon termed preferred orientation, limiting our ability to obtain 3-D reconstructions. Surfactants are often used as sample additives to prevent AWI-induced deterioration, but no general strategy exists for surfactant choice, requiring laborious screening for each sample. To circumvent these issues, we developed SurfACT, a cocktail of diverse surfactants with distinct physicochemical properties that limits AWI-dependent sample denaturation and orientation bias, while mitigating individual surfactant-specific drawbacks. Here we demonstrate SurfACTs effectiveness with four proteins plagued by AWI-induced issues, including two species of hemagglutinin (HA), molybdenum-iron protein (MoFeP) from the nitrogenase enzyme, and aldolase. All four samples show drastically improved viewing distribution and map completeness when SurfACT is applied. Cryogenic electron tomography demonstrates that SurfACT redistributes particles from the AWI into the bulk ice, driving signal recovery and inhibiting denaturation. This versatile sample additive minimizes sample-specific screening and expands the capabilities and range of suitable samples for cryoEM.

biophysics↗

bioRxiv2025

Tryptoline Stereoprobe Elaboration Identifies Inhibitors of the GRPEL1-HSPA9 Chaperone Complex

Activity-based protein profiling has identified hundreds of proteins from diverse classes that react at specific cysteine residues with stereochemically defined electrophilic compounds (stereoprobes) in human cells. The structure-activity relationships underlying these stereoprobe-protein interactions, however, remain poorly understood. Here we show that the protein interaction landscape of tryptoline acrylamide stereoprobes can be profoundly altered by structural modifications distal to the acrylamide reactive group. The majority of stereoprobe liganding events occurred at non-orthosteric sites and mostly evaded assignment by the machine learning-based co-folding model Boltz-2, which instead tended to misplace the stereoprobes in orthosteric pockets (an outcome we term "orthostery burnout"). We found that stereoprobes reacting with C124 in the nucleotide exchange factor GRPEL1 disrupt interactions with the mitochondrial HSP70 chaperone HSPA9/mortalin, leading to impairments in mitochondrial protein import and induction of mitophagy. Our results highlight tryptoline acrylamides as a versatile source of covalent ligands targeting non-orthosteric sites on proteins, including tool compounds that perturb the mitochondrial HSP70 chaperone system.

biochemistry↗

bioRxiv2025

ANTIDOTE: A Metadata-Driven Neural Network for Improving CryoEM 3-D Particle Sorting

Despite the maturation of cryogenic electron microscopy (cryoEM) methodologies, generating high-resolution three-dimensional (3-D) reconstructions from micrographs is a time-intensive process involving iterative rounds of subjective data curation and hyperparameter optimization. Current approaches to particle classification are often unable to remove all low-quality particles from particle stacks, largely due to the low signal-to-noise ratio, the high dimensionality of particle images, and the multiple degrees of freedom associated with each particles unknown rotation, orientation, and class assignment. The retention of low-quality particles negatively affects the overall quality of the final EM density and continued efforts to eliminate their deleterious contributions are warranted. Here, we present ANTIDOTE (A Neural network Trained In Deleterious Object deTection and Elimination), a neural network framework that discriminates between constructive and deleterious particles using per-particle metadata generated during 3-D classification in RELION. Using benchmark and real-world cryoEM datasets, we demonstrate that ANTIDOTE paired with RELION 3-D classification achieves higher particle classification accuracy than conventional data processing approaches alone, yielding improvements in reconstruction quality, global and local resolution, and map interpretability while reducing time-consuming hyperparameter optimization. We additionally detail practical use-case scenarios for ANTIDOTE and demonstrate its versatility in increasing particle curation accuracy for high-quality cryoEM reconstruction.

biophysics↗

bioRxiv2025

An allosteric network governs Tom70 conformational dynamics to coordinate mitochondrial import

Tom70 mediates mitochondrial protein import by coordinating the transfer of cytosolic preproteins from Hsp70/Hsp90 to the translocase of the outer membrane (TOM) complex. In humans, the cytosolic domain of Tom70 (HsTom70c) is entirely -helical and comprises modular TPR motifs divided into an N-terminal chaperone-binding domain (NTD) and a C-terminal preprotein-binding domain (CTD). However, the mechanisms linking these functional regions remain poorly understood. Here, we present the 2.04 [A] crystal structure of unliganded HsTom70c, revealing two distinct conformations - open and closed - within the asymmetric unit. These states are stabilized in part by interdomain crystal contacts and are supported in solution by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Principal component and dynamical network analyses reveal a continuum of motion linking the NTD and CTD via key structural elements, notably residues in helices 7, 8, and 25. Engagement of the CTD by the viral protein Orf9b interrupts this network, stabilizing a partially-closed intermediate conformation and dampening dynamics at distal NTD sites. Collectively, our findings lay the groundwork for understanding Tom70 allostery and provide a framework for dissecting its mechanistic roles in chaperone engagement, mitochondrial import, and viral subversion.

biophysics↗

bioRxiv2025

Short 5' UTRs serve as a marker for viral mRNA translation inhibition by the IFIT2-IFIT3 antiviral complex

Recognition of "non-self" nucleic acids, including cytoplasmic dsDNA, dsRNA, or mRNAs lacking proper 5 cap structures, is critical for the innate immune response to viruses. Here, we demonstrate that short 5 untranslated regions (UTRs), a characteristic of many viral mRNAs, can also serve as a molecular pattern for innate immune recognition via the interferon-induced proteins IFIT2 and IFIT3. The IFIT2-IFIT3 heterodimer, formed through an intricate domain swap structure resolved by cryo-EM, mediates viral mRNA 5 end recognition, translation inhibition, and ultimately antiviral activity. Critically, 5 UTR lengths <50 nucleotides are necessary and sufficient to sensitize an mRNA to translation inhibition by the IFIT2-IFIT3 complex. Accordingly, diverse viruses whose mRNAs contain short 5 UTRs, such as vesicular stomatitis virus and parainfluenza virus 3, are sensitive to IFIT2-IFIT3-mediated antiviral activity. Our work thus reveals a pattern of antiviral nucleic acid immune recognition that takes advantage of the inherent constraints on viral genome size.

microbiology↗

bioRxiv2024

Capturing the Conformational Heterogeneity of HSPB1 Chaperone Oligomers at Atomic Resolution

Small heat shock proteins (sHSPs), including HSPB1, are essential regulators of cellular proteostasis that interact with unfolded and partially folded proteins to prevent aberrant misfolding and aggregation. These proteins fulfill a similar role in biological condensates, where they interact with intrinsically disordered proteins to modulate their liquid-liquid and liquid-to-solid phase transitions. Characterizing sHSP structure, dynamics, and client interactions is challenging due to their partially disordered nature, their tendency to form polydisperse oligomers, and their diverse range of clients. In this work, we leverage various biophysical methods, including fast 1H-based magic angle spinning (MAS) NMR spectroscopy, molecular dynamics (MD) simulations and modeling, to shed new light on the structure and dynamics of HSPB1 oligomers. Using split-intein mediated segmental labeling, we provide unambiguous evidence that in the oligomer context the N-terminal domain (NTD) of HSPB1 is rigid and adopts an ensemble of heterogenous conformations, the -crystallin domain (ACD) forms dimers and experiences multiple distinct local environments, while the C-terminal domain (CTD) remains highly dynamic. Our computational models suggest that the NTDs participate in extensive NTD-NTD and NTD-ACD interactions and are sequestered within the oligomer interior. We further demonstrate that HSPB1 higher order oligomers disassemble into smaller oligomeric species in the presence of a client protein and that an accessible NTD is essential for HSPB1 partitioning into condensates and interactions with client proteins. Our integrated approach provides a high-resolution view of the complex oligomeric landscape of HSPB1 and sheds light on the elusive network of interactions that underly HSPB1 function in biological condensates. Significance statementHSPB1 is a ubiquitous cellular chaperone that helps prevent the aberrant aggregation of intrinsically disordered proteins involved in biological condensates and neurodegenerative diseases. Despite its central role in this process, many aspects of HSPB1s structure and interactions with clients are not well understood due to its tendency to form polydisperse oligomeric structures and to function in heterogeneous condensate environments. Here, we present an integrated approach that includes segmental labeling, fast MAS NMR spectroscopy, and computational tools to characterize the structure and dynamics of HSPB1 in its oligomeric form and within client condensates at high resolution. Our approach, which is applicable to other complex biological systems, highlights the important role of HSPB1s N-terminal domain in oligomeric assembly and interactions with clients.

biophysics↗

bioRxiv2024

Preparation of oxygen-sensitive proteins for high-resolution cryoEM structure determination using (an)aerobic blot-free vitrification

High-quality grid preparation for single-particle cryogenic electron microscopy (cryoEM) remains a bottleneck for routinely obtaining high-resolution structures. The issues that arise from traditional grid preparation workflows are particularly exacerbated for oxygen-sensitive proteins, including metalloproteins, whereby oxygen-induced damage and alteration of oxidation states can result in protein inactivation, denaturation, and/or aggregation. Indeed, 99% of the current structures in the EMBD were prepared aerobically and limited successes for anaerobic cryoEM grid preparation exist. Current practices for anaerobic grid preparation involve a vitrification device located in an anoxic chamber, which presents significant challenges including temperature and humidity control, optimization of freezing conditions, costs for purchase and operation, as well as accessibility. Here, we present a streamlined approach that allows for the (an)aerobic vitrification of oxygen-sensitive proteins using an automated aerobic blot-free grid vitrification device - the SPT Labtech chameleon. This robust workflow allows for high-resolution structure determination of dynamic, oxygen-sensitive proteins, of varying complexity and molecular weight.

biophysics↗

bioRxiv2024

Structural insights into GrpEL1-mediated nucleotide and substrate release of human mitochondrial Hsp70

Maintenance of protein homeostasis is necessary for cell viability and depends on a complex network of chaperones and co-chaperones, including the heat-shock protein 70 (Hsp70) system. In human mitochondria, mitochondrial Hsp70 (mortalin) and the nucleotide exchange factor (GrpEL1) work synergistically to stabilize proteins, assemble protein complexes, and facilitate protein import. However, our understanding of the molecular mechanisms guiding these processes is hampered by limited structural information. To elucidate these mechanistic details, we used cryoEM to determine the first structures of full-length human mortalin-GrpEL1 complexes in previously unobserved states. Our structures and molecular dynamics simulations allow us to delineate specific roles for mortalin-GrpEL1 interfaces and to identify steps in GrpEL1-mediated nucleotide and substrate release by mortalin. Subsequent analyses reveal conserved mechanisms across bacteria and mammals and facilitate a complete understanding of sequential nucleotide and substrate release for the Hsp70 chaperone system.

biophysics↗

bioRxiv2024

Tuning ice thickness using the chameleon for high-quality cryoEM data collection

Advances in single-particle cryogenic electron microscopy (cryoEM) now allow for routine structure determination of well-behaved biological specimens to high-resolution. Despite advances in the electron microscope, direct electron detectors, and data processing software, the preparation of high-quality grids with thin layers of vitreous ice containing the specimen of interest in random orientations remains a critical bottleneck for many projects. Although numerous efforts have been dedicated to overcoming hurdles frequently encountered during specimen vitrification using traditional blot-and-plunge specimen preparation techniques, the development of blot-free grid preparation devices provide a unique opportunity to carefully tune ice thickness, particle density, and specimen behavior during the vitrification process for improvements in image quality. Here, we describe critical steps of high-quality grid preparation using a SPT Labtech chameleon, evaluation of grid quality/ice thickness using the chameleon software, high-throughput imaging in the electron microscope, and recommend steps for troubleshooting grid preparation when standard parameters fail to yield suitable specimen. Video LinkContents of this manuscript are available as a video tutorial. This video can be found here

biophysics↗

bioRxiv2022

CryoEM Structures of the Nitrogenase Complex During Catalytic Turnover

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multi-electron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here we report the cryogenic electron microscopic interrogation of the nitrogenase complex under enzymatic turnover conditions, which has enabled the structural characterization of the nitrogenase reaction intermediates at high resolution for the first time. Our structures show that asymmetry governs all aspects of nitrogenase mechanism including ATP hydrolysis, protein-protein interactions, and catalysis. Furthermore, they reveal several previously unobserved, mechanistically relevant conformational changes near the catalytic iron-molybdenum cofactor that are correlated with the nucleotide-hydrolysis state of the enzyme. One-sentence summaryHigh-resolution cryoEM structures of the nitrogenase complex obtained under turnover provide new mechanistic insights.

biochemistry↗

bioRxiv2020

Outcomes of the 2019 EMDataResource model challenge: validation of cryo-EM models at near-atomic resolution

This paper describes outcomes of the 2019 Cryo-EM Map-based Model Metrics Challenge sponsored by EMDataResource (www.emdataresource.org). The goals of this challenge were (1) to assess the quality of models that can be produced using current modeling software, (2) to check the reproducibility of modeling results from different software developers and users, and (3) compare the performance of current metrics used for evaluation of models. The focus was on near-atomic resolution maps with an innovative twist: three of four target maps formed a resolution series (1.8 to 3.1 [A]) from the same specimen and imaging experiment. Tools developed in previous challenges were expanded for managing, visualizing and analyzing the 63 submitted coordinate models, and several novel metrics were introduced. The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual laboratory experiments and holdings of structure data archives such as the Protein Data Bank. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived from these benchmark maps by 13 participating teams, representing both widely used and novel modeling approaches. We also evaluate the pros and cons of the commonly used metrics to assess model quality and recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed density in the cryo-EM map.

biochemistry↗

bioRxiv2019

Sub-2 A Resolution Structure Determination Using Single-Particle Cryo-EM at 200 keV

Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination of well-behaved specimens to high-resolution, there nonetheless remains a discrepancy between the resolutions attained for biological specimens and the information limits of modern transmission electron microscopes (TEMs). Instruments operating at 300 kV equipped with DEDs are the current paradigm for high-resolution single-particle cryo-EM, while 200 kV TEMs remain comparatively underutilized for purposes beyond sample screening. Here, we expand upon our prior work and demonstrate that one such 200 kV microscope, the Talos Arctica, equipped with a K2 DED is capable of determining structures of macromolecules to as high as [~]1.7 [A] resolution. At this resolution, ordered water molecules are readily assigned and holes in aromatic residues can be clearly distinguished in the reconstructions. This work emphasizes the utility of 200 keV for high-resolution single-particle cryo-EM and applications such as structure-based drug design.

biophysics↗

bioRxiv2018

High-resolution structure determination of sub-100 kilodalton complexes using conventional cryo-EM

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our [~]2.9 [A] structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our [~]2.8 [A] and [~]3.2 [A] resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.

biophysics↗

bioRxiv2018

Conformational ensemble of the human TRPV3 ion channel

Transient receptor potential vanilloid channel 3 (TRPV3), a member of the thermosensitive TRP (thermoTRPV) channels, is activated by warm temperatures and serves as a key regulator of normal skin physiology through the release of pro-inflammatory messengers. Mutations in trpv3 have been identified as the cause of the congenital skin disorder, Olmsted syndrome. Unlike other members of the thermoTRPV channel family, TRPV3 sensitizes upon repeated simulation, yet a lack of structural information about the channel precludes a molecular-level understanding of TRPV3 sensitization and gating. Here, we present the cryo-electron microscopy structures of apo and sensitized human TRPV3, as well as several structures of TRPV3 in the presence of the common thermoTRPV agonist 2-aminoethoxydiphenyl borate (2-APB). Our results show -to-{pi}-helix transitions in the S6 during sensitization, and suggest a critical role for the S4-S5 linker {pi}-helix during ligand gating.

biochemistry↗

bioRxiv2017

Achieving Better Than 3 A Resolution By Single Particle Cryo-EM At 200 keV

Technical and methodological advances in single-particle cryo-electron microscopy (cryo-EM) have expanded the technique into a resolution regime that was previously only attainable by X-ray crystallography. Although single-particle cryo-EM has proven to be a useful technique for determining the structures of biomedically relevant molecules at near-atomic resolution, nearly 98% of the structures resolved to better than 4 [A] resolution have been determined using 300 keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain cryo-EM reconstructions of macromolecular complexes at a range of sizes to better than 3 [A] resolution using a 200 keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules, features previously thought only to be resolvable using TEMs operating at 300 keV.

biophysics↗

bioRxiv2017

A multi-model approach to assessing local and global cryo-EM map quality

There does not currently exist a standardized indicator of how well a cryo-EM-derived model represents the density from which it was generated. We present a straightforward methodology that utilizes freely available tools to generate a suite of independent models and to evaluate their convergence in an EM density. These analyses provide both a quantitative and qualitative assessment of the precision of the models and their representation of the density, respectively, while concurrently providing a platform for assessing both global and local EM map quality. We further use standardized datasets to provide an expected model-model agreement criterion for EM maps reported to be at 5 [A] resolution or better. Associating multiple atomic models with a deposited EM map provides a rapid and accessible reporter of convergence, a strong indicator of highly resolved molecular detail, and is an important step toward an FSC-independent assessment of map and model quality.

biophysics↗