Cryo-electron Microscopy Research Articles

Nucleosome flipping drives kinetic proofreading and processivity by SWR1.

The yeast SWR1 complex catalyses the exchange of histone H2A-H2B dimers in nucleosomes, with Htz1-H2B dimers1-3. Here we used single-molecule analysis to demonstrate two-step double exchange of the two H2A-H2B dimers in a canonical yeast nucleosome with Htz1-H2B dimers, and showed that double exchange can be processive without release of the nucleosome from the SWR1 complex. Further analysis showed that bound nucleosomes flip between two states, with each presenting a different face, and hence histone dimer, to SWR1. The bound dwell time is longer when an H2A-H2B dimer is presented for exchange than when presented with an Htz1-H2B dimer. A hexasome intermediate in the reaction is bound to the SWR1 complex in a single orientation with the 'empty' site presented for dimer insertion. Cryo-electron microscopy analysis revealed different populations of complexes showing nucleosomes caught 'flipping' between different conformations without release, each placing a different dimer into position for exchange, with the Swc2 subunit having a key role in this process. Together, the data reveal a processive mechanism for double dimer exchange that explains how SWR1 can 'proofread' the dimer identities within nucleosomes.

Site-Specific Histidine Aza-Michael Addition in Proteins Enabled by a Ferritin-Based Metalloenzyme.

Histidine modifications of proteins are broadly based on chemical methods triggering N-substitution reactions such as aza-Michael addition at histidine's moderately nucleophilic imidazole side chain. While recent studies have demonstrated chemoselective, histidine-specific modifications by further exploiting imidazole's electrophilic reactivity to overcome interference from the more nucleophilic lysine and cysteine, achieving site-specific histidine modifications remains a major challenge due to the absence of spatial control over chemical processes. Herein, through X-ray crystallography and cryo-electron microscopy structural studies, we describe the rational design of a nature-inspired, noncanonical amino-acid-incorporated, human ferritin-based metalloenzyme that is capable of introducing site-specific post-translational modifications (PTMs) to histidine in peptides and proteins. Specifically, chemoenzymatic aza-Michael additions on single histidine residues were carried out on eight protein substrates ranging from 10 to 607 amino acids including the insulin peptide hormone. By introducing an insulin-targeting peptide into our metalloenzyme, we further directed modifications to be carried out site-specifically on insulin's B-chain histidine 5. The success of this biocatalysis platform outlines a novel approach in introducing residue- and, moreover, site-specific post-translational modifications to peptides and proteins, which may further enable reactions to be carried out in vivo.

Tubulin acetyltransferases access and modify the microtubule luminal K40 residue through anchors in taxane-binding pockets.

Acetylation at α-tubulin K40 is the sole post-translational modification preferred to occur inside the lumen of hollow cylindrical microtubules. However, how tubulin acetyltransferases access the luminal K40 in micrometer-long microtubules remains unknown. Here, we use cryo-electron microscopy and single-molecule reconstitution assays to reveal the enzymatic mechanism for tubulin acetyltransferases to modify K40 in the lumen. One tubulin acetyltransferase spans across the luminal lattice, with the catalytic core docking onto two α-tubulins and the enzyme's C-terminal domain occupying the taxane-binding pockets of two β-tubulins. The luminal accessibility and enzyme processivity of tubulin acetyltransferases are inhibited by paclitaxel, a microtubule-stabilizing chemotherapeutic agent. Characterizations using recombinant tubulins mimicking preacetylated and postacetylated K40 show the crosstalk between microtubule acetylation states and the cofactor acetyl-CoA in enzyme turnover. Our findings provide crucial insights into the conserved multivalent interactions involving α- and β-tubulins to acetylate the confined microtubule lumen.

The structures of protein kinase A in complex with CFTR: Mechanisms of phosphorylation and noncatalytic activation

Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating numerous substrates, including ion channels, enzymes, and transcription factors. It has long served as a model system for understanding the eukaryotic kinases. Using cryoelectron microscopy, we present complex structures of the PKA catalytic subunit (PKA-C) bound to a full-length protein substrate, the cystic fibrosis transmembrane conductance regulator (CFTR)-an ion channel vital to human health. CFTR gating requires phosphorylation of its regulatory (R) domain. Unphosphorylated CFTR engages PKA-C at two locations, establishing two "catalytic stations" near to, but not directly involving, the R domain. This configuration, coupled with the conformational flexibility of the R domain, permits transient interactions of the eleven spatially separated phosphorylation sites. Furthermore, we determined two structures of the open-pore CFTR stabilized by PKA-C, providing a molecular basis to understand how PKA-C stimulates CFTR currents even in the absence of phosphorylation.

Genetically Engineered Liposwitch-Based Nanomaterials.

Fusion of intrinsically disordered and globular proteins is a powerful strategy to create functional nanomaterials. However, the immutable nature of genetic encoding restricts the dynamic adaptability of nanostructures postexpression. To address this, we envisioned using a myristoyl switch, a protein that combines allostery and post-translational modifications─two strategies that modify protein properties without altering their sequence─to regulate intrinsically disordered protein (IDP)-driven nanoassembly. A typical myristoyl switch, allosterically activated by a stimulus, reveals a sequestered lipid for membrane association. We hypothesize that this conditional exposure of lipids can regulate the assembly of fusion proteins, a concept we term "liposwitching". We tested this by fusing recoverin, a calcium-dependent myristoyl switch, with elastin-like polypeptide, a thermoresponsive model IDP. Biophysical analyses confirmed recoverin's myristoyl-switch functionality, while dynamic light scattering and cryo-transmission electron microscopy showed distinct calcium- and lipidation-dependent phase separation and assembly. This study highlights liposwitching as a viable strategy for controlling DP-driven nanoassembly, enabling applications in synthetic biology and cellular engineering.

Aggregation Dynamics of a 150 kDa Aβ42 Oligomer: Insights from Cryo Electron Microscopy and Multimodal Analysis

Protein misfolding is a widespread phenomenon that can result in the formation of protein aggregates, which are markers of various disease states, including Alzheimer's disease (AD). In AD, amyloid beta (Aβ) peptides are key players in the disease's progression, particularly the 40- and 42 residue variants, Aβ40 and Aβ42. These peptides aggregate to form amyloid plaques and contribute to neuronal toxicity. Recent research has shifted attention from solely Aβ fibrils to also include Aβ protofibrils and oligomers as potentially critical pathogenic agents. Particularly, oligomers demonstrate more significant toxicity compared to other Aβ specie. Hence, there is an increased interest in studying the correlation between toxicity and their structure and aggregation pathway. The present study investigates the aggregation of a 150kDa Aβ42 oligomer that does not lead to fibril formation. Using negative stain transmission electron microscopy (TEM), size exclusion chromatography (SEC), dynamic light scattering (DLS), and cryo-electron microscopy (cryo-EM), we demonstrate that 150kDa Aβ42 oligomers form higher-order string-like assemblies over time. These strings are unique from the classical Aβ fibrils. The significance of our work lies in elucidating molecular behavior of a novel non-fibrillar form of Aβ42 aggregate.

Challenge for Deep Learning: Protein Structure Prediction of Ligand-Induced Conformational Changes at Allosteric and Orthosteric Sites.

In the realm of biomedical research, understanding the intricate structure of proteins is crucial, as these structures determine how proteins function within our bodies and interact with potential drugs. Traditionally, methods like X-ray crystallography and cryo-electron microscopy have been used to unravel these structures, but they are often challenging, time-consuming and costly. Recently, a breakthrough in computational biology has emerged with the development of deep learning algorithms capable of predicting protein structures based on their amino acid sequences (Jumper, J., et al. Nature 2021, 596, 583. Lane, T. J. Nature Methods 2023, 20, 170. Kryshtafovych, A., et al. Proteins: Structure, Function and Bioinformatics 2021, 89, 1607). This study focuses on predicting the dynamic changes that proteins undergo upon ligand binding, specifically when they bind to allosteric sites, i.e. a pocket different from the active site. Allosteric modulators are particularly important for drug discovery, as they open new avenues for designing drugs that can target proteins more effectively and with fewer side effects (Nussinov, R.; Tsai, C. J. Cell 2013, 153, 293). To study this, we curated a data set of 578 X-ray structures comprised of proteins displaying orthosteric and allosteric binding as well as a general framework to evaluate deep learning-based structure prediction methods. Our findings demonstrate the potential and current limitations of deep learning methods, such as AlphaFold2 (Jumper, J., et al. Nature 2021, 596, 583), NeuralPLexer (Qiao, Z., et al. Nat Mach Intell 2024, 6, 195), and RoseTTAFold All-Atom (Krishna, R., et al. Science 2024, 384, eadl2528) to predict not just static protein structures but also the dynamic conformational changes. Herein we show that predicting the allosteric induce-fit conformation still poses a challenge to deep learning methods as they more accurately predict the orthosteric bound conformation compared to the allosteric induce fit conformation. For AlphaFold2, we observed that conformational diversity, and sampling between the apo and holo state could be increased by modifying the MSA depth, but this did not enhance the ability to generate conformations close to the allosteric induced-fit conformation. To further support advancements in protein structure prediction field, the curated data set and evaluation framework are made publicly available.

Structures and pH-dependent dimerization of the sevenless receptor tyrosine kinase

Sevenless (Sev) is a Drosophila receptor tyrosine kinase (RTK) required for the specification of the R7 photoreceptor. It is cleaved into α and β subunits and binds the ectodomain of the G-protein-coupled receptor bride of sevenless (Boss). Previous work showed that the Boss ectodomain could bind but not activate Sev; rather, the whole seven-pass transmembrane Boss was required. Here, we show that Sev does not need to be cleaved to function and that a single-pass transmembrane form of Boss activates Sev. We use cryo-electron microscopy and biophysical methods to determine the structural basis of ligand binding and pH-dependent dimerization of Sev, and we discuss the implications in the process of Sev activation. The Sev human homolog, receptor oncogene from sarcoma 1 (ROS1), is associated with oncogenic transformations, and we discuss their structural similarities.

Isolation and escape mapping of broadly neutralizing antibodies against emerging delta-coronaviruses.

Porcine delta-coronavirus (PDCoV) spillovers were recently detected in febrile children, underscoring the recurrent zoonoses of divergent CoVs. To date, no vaccines or specific therapeutics are approved for use in humans against PDCoV. To prepare for possible future PDCoV epidemics, we isolated PDCoV spike (S)-directed monoclonal antibodies (mAbs) from humanized mice and found that two, designated PD33 and PD41, broadly neutralized a panel of PDCoV variants. Cryoelectron microscopy (cryo-EM) structures of PD33 and PD41 in complex with the S receptor-binding domain (RBD) and ectodomain trimer revealed the epitopes recognized by these mAbs, rationalizing their broad inhibitory activity. We show that both mAbs competitively interfere with host aminopeptidase N binding to neutralize PDCoV and used deep-mutational scanning epitope mapping to associate RBD antigenic sites with mAb-mediated neutralization potency. Our results indicate a PD33-PD41 mAb cocktail may heighten the barrier to escape. PD33 and PD41 are candidates for clinical advancement against future PDCoV outbreaks.

STK19 drives transcription-coupled repair by stimulating repair complex stability, RNA Pol II ubiquitylation, and TFIIH recruitment.

Transcription-coupled nucleotide excision repair (TC-NER) efficiently eliminates DNA damage that impedes gene transcription by RNA polymerase II (RNA Pol II). TC-NER is initiated by the recognition of lesion-stalled RNA Pol II by CSB, which recruits the CRL4CSA ubiquitin ligase and UVSSA. RNA Pol II ubiquitylation at RPB1-K1268 by CRL4CSA serves as a critical TC-NER checkpoint, governing RNA Pol II stability and initiating DNA damage excision by TFIIH recruitment. However, the precise regulatory mechanisms of CRL4CSA activity and TFIIHrecruitment remain elusive. Here, we reveal human serine/threonine-protein kinase 19 (STK19) as a TC-NER factor, which is essential for correct DNA damage removal and subsequent transcription restart. Cryogenic electron microscopy (cryo-EM) studies demonstrate that STK19 is an integral part of the RNA Pol II-TC-NER complex, bridging CSA, UVSSA, RNA Pol II, and downstream DNA. STK19 stimulates TC-NER complex stability and CRL4CSA activity, resulting in efficient RNA Pol II ubiquitylation and correct UVSSA and TFIIH binding. These findings underscore the crucial role of STK19 as a core TC-NER component.

Lipid nanoparticle properties explored using online asymmetric flow field-flow fractionation coupled with small angle X-ray scattering: Beyond average characterisation

This study employs asymmetric flow field-flow fractionation online coupled with small angle X-ray scattering at a synchrotron beamline, along with multiple downstream detectors, including multi-angle light scattering, dual wavelength UV and dRI. This setup enables size-resolved characterization of lipid nanoparticles, allowing for a detailed comparison between empty and cargo-loaded lipid nanoparticles intended for nucleic acid delivery. Batch-mode characterization techniques, including cryogenic transmission electron microscopy and dynamic light scattering, alongside collection of fractions for offline characterization with liquid chromatography-charged aerosol detection, allowed for determination of the particle morphology, hydrodynamic radius, and the lipid composition over the size distribution. Cargo-containing and empty lipid nanoparticles show differences in density, and loaded particles exhibit a broader size distribution and a higher frequency of blebs at the surface. Both samples consist of spherical core–shell structured particles, with no distinguishable internal structure. A pivotal finding, often assumed until now, is that the mole fraction of each individual lipid component closely matches the original formulation. This work contributes to a more detailed understanding of lipid nanoparticles, supporting their continued development and rational design in medical applications.

Perspectives Toward an Integrative Structural Biology Pipeline With Atomic Force Microscopy TopographicImages.

After the recent double revolutions in structural biology, which include the use of direct detectors for cryo-electron microscopy resulting in a significant improvement in the expected resolution of large macromolecule structures, and the advent of AlphaFold which allows for near-accurate prediction of any protein structures, the field of structural biology is now pursuing more ambitious targets, including several MDa assemblies. But complex target systems cannot be tackled using a single biophysical technique. The field of integrative structural biology has emerged as a global solution. The aim is to integrate data from multiple complementary techniques to produce a final three-dimensional model that cannot be obtained from any single technique. The absence of atomic force microscopy data from integrative structural biology platforms is not necessarily due to its nm resolution, as opposed to Å resolution for x-ray crystallography, nuclear magnetic resonance, or electron microscopy. Rather a significant issue was that the AFM topographic data lacked interpretability. Fortunately, with the introduction of the AFM-Assembly pipeline and other similar tools, it is now possible to integrate AFM topographic data into integrative modeling platforms. The advantages of single molecule techniques, such as AFM, include the ability to confirm experimentally any assembled molecular models or to produce alternative conformations that mimic the inherent flexibility of large proteins or complexes. The review begins with a brief overview of the historical developments of AFM data in structural biology, followed by an examination of the strengths and limitations of AFM imaging, which have hindered its integration into modern modeling platforms. This review discusses the correction and improvement of AFM topographic images, as well as the principles behind the AFM-Assembly pipeline. It also presents and discusses a series of challenges that need to be addressed in order to improve the incorporation of AFM data into integrative modeling platform.

Amyloid fibril structures and ferroptosis activation induced by ALS-causing SOD1 mutations.

Over 200 genetic mutations in copper-zinc superoxide dismutase (SOD1) have been linked to amyotrophic lateral sclerosis (ALS). Among these, two ALS-causing mutants, histidine-46→arginine (H46R) and glycine-85→arginine (G85R), exhibit a decreased capacity to bind metal ions. Here, we report two cryo-electron microscopy structures of amyloid fibrils formed by H46R and G85R. These mutations lead to the formation of amyloid fibrils with unique structures distinct from those of the native fibril. The core of these fibrils features a serpentine arrangement with seven or eight β strands, secured by a hydrophobic cavity and a salt bridge between arginine-85 and aspartic acid-101 in the G85R fibril. We demonstrate that these mutant fibrils are notably more toxic and capable of promoting the aggregation of wild-type SOD1 more effectively, causing mitochondrial impairment and activating ferroptosis in cell cultures, compared to wild-type SOD1 fibrils. Our study provides insights into the structural mechanisms by which SOD1 mutants aggregate and induce cytotoxicity in ALS.

Fully human monoclonal antibody targeting the cysteine-rich substrate-interacting region of ADAM17 on cancer cells

ADAM17 sheds EGFR/erbB ligands and triggers oncogenic pathways that lead to the progression of solid tumors. We targeted the ADAM17 disintegrin and cysteine rich domain region (D+C) to generate a panel of single-chain antibody fragments (scFvs) that selectively bind to the D or C domains of ADAM17, but not of ADAM10 or ADAM19. From the panel, we selected one scFv, referred to as C12, based on its high binding affinity towards the target, and re-formatted it to a full IgG for further studies. High-resolution cryo-electron microscopy studies documented that the mAb binds to the ADAM17 C-domain that in ADAM proteases, notably ADAM10 and ADAM17, is known to impart substrate-specificity. The C12 mAb significantly inhibited EGFR phosphorylation in cancer cell lines by hindering the cleavage of EGFR ligands tethered to the cell surface. This inhibition provides a mechanism for potential anti-tumor effects, and indeed C12 diminished the viability of a variety of EGFR-expressing cancer cell lines. Cell-based ELISA studies revealed that C12 preferentially bound to activated ADAM17 present on tumor cells, as compared to the autoinhibited ADAM17 that is the predominant form on HEK293 and other non-tumor cells. C12 also exhibited tumor growth inhibition in an ovarian cancer xenograft mouse model. Consistent with its selective tumor cell binding in vitro, radioimmuno PET (positron emission tomography) imaging with 89Zr-DFO-C12 in mouse xenograft models confirmed tumoral accumulation of the C12 mAb.

Crystal structure of the alternative complex III from the phototrophic bacterium Chloroflexus aurantiacus.

Alternative complex III (ACIII) is a multi-subunit quinol:electron acceptor oxidoreductase that couples quinol oxidation with transmembrane proton translocation in bacterial respiratory and photosynthetic electron transport chains. Four ACIII cryoelectron microscopy (cryo-EM) structures are known. However, the effects of cryo-EM versus X-ray crystallography structure determination on ACIII structure are unclear. Here, we report a 3.25Å crystal structure of photosynthetic ACIII from Chloroflexus aurantiacus (CaACIIIp), revealing eight subunits (ActA-G and I) with four iron-sulfur clusters and six c-type hemes, a menaquinol-binding site, and two proton translocation passages. Structural comparisons with the previously reported cryo-EM structures reveal slight local conformational changes in the solvent-exposed regions of ActB, ActD, ActG, and the transmembrane (TM) helix of subunit I. The regions conferring structural flexibility possess low sequence conservation across species. However, the core functional modules containing the menaquinol-binding pocket, redox centers, and proton translocation passages remain unchanged, preserving the enzyme's activity.

Impact of Dilute DIO Additive on Local Microstructure of Fluorinated, pNDI-Based Polymer Solar Cells.

The performance of all-polymer solar cells is often enhanced by incorporating solvent additives during solution processing. In particular, blends based on the model all-polymer system PBDBT:N2200 have been shown to have increased short-circuit current and fill factor when processed with dilute diiodooctane (DIO). However, the morphological mechanism that drives the increase in performance is often not well understood due to limitations in common characterization techniques. In this study, it is shown that a combination of X-ray techniques with cryogenic high-resolution transmission electron microscopy (HRTEM) analysis can provide a quantitative and spatially resolved picture of polymer chain orientation and alignment in all-polymer blends. It is found that DIO induces vertical phase separation in PBDBT-2F:F-N2200 and increases donor crystallite thickness in the pi-stacking direction leading to an acceptor-rich film surface. However, it is also shown that DIO does not disrupt the formation of face-on donor-acceptor interfaces. These findings suggest that dilute DIO primarily affects crystalline domain formation in single component regions as opposed to mixed regions; thus, dilute DIO can impact vertical charge transport pathways without sacrificing donor-acceptor interfacial connectivity.

Structural basis for molecular assembly of fucoxanthin chlorophyll a/c-binding proteins in a diatom photosystem I supercomplex

Photosynthetic organisms exhibit remarkable diversity in their light-harvesting complexes (LHCs). LHCs are associated with photosystem I (PSI), forming a PSI-LHCI supercomplex. The number of LHCI subunits, along with their protein sequences and pigment compositions, has been found to differ greatly among the PSI-LHCI structures. However, the mechanisms by which LHCIs recognize their specific binding sites within the PSI core remain unclear. In this study, we determined the cryo-electron microscopy structure of a PSI supercomplex incorporating fucoxanthin chlorophyll a/c-binding proteins (FCPs), designated as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana CCMP1335. Structural analysis of PSI-FCPI revealed five FCPI subunits associated with a PSI monomer; these subunits were identified as RedCAP, Lhcr3, Lhcq10, Lhcf10, and Lhcq8. Through structural and sequence analyses, we identified specific protein–protein interactions at the interfaces between FCPI and PSI subunits, as well as among FCPI subunits themselves. Comparative structural analyses of PSI-FCPI supercomplexes, combined with phylogenetic analysis of FCPs from T. pseudonana and the diatom Chaetoceros gracilis, underscore the evolutionary conservation of protein motifs crucial for the selective binding of individual FCPI subunits. These findings provide significant insights into the molecular mechanisms underlying the assembly and selective binding of FCPIs in diatoms.

Structural basis for molecular assembly of fucoxanthin chlorophyll a/c-binding proteins in a diatom photosystem I supercomplex.

Photosynthetic organisms exhibit remarkable diversity in their light-harvesting complexes (LHCs). LHCs are associated with photosystem I (PSI), forming a PSI-LHCI supercomplex. The number of LHCI subunits, along with their protein sequences and pigment compositions, has been found to differ greatly among the PSI-LHCI structures. However, the mechanisms by which LHCIs recognize their specific binding sites within the PSI core remain unclear. In this study, we determined the cryo-electron microscopy structure of a PSI supercomplex incorporating fucoxanthin chlorophyll a/c-binding proteins (FCPs), designated as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana CCMP1335. Structural analysis of PSI-FCPI revealed five FCPI subunits associated with a PSI monomer; these subunits were identified as RedCAP, Lhcr3, Lhcq10, Lhcf10, and Lhcq8. Through structural and sequence analyses, we identified specific protein-protein interactions at the interfaces between FCPI and PSI subunits, as well as among FCPI subunits themselves. Comparative structural analyses of PSI-FCPI supercomplexes, combined with phylogenetic analysis of FCPs from T. pseudonana and the diatom Chaetoceros gracilis, underscore the evolutionary conservation of protein motifs crucial for the selective binding of individual FCPI subunits. These findings provide significant insights into the molecular mechanisms underlying the assembly and selective binding of FCPIs in diatoms.

Activity and cryo-EM structure of the polymerase domain of the human norovirus ProPol precursor.

Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor and mature proteins during processing of the viral polyprotein that are essential to the viral lifecycle. One such precursor is protease-polymerase (ProPol), a multi-functional enzyme comprised of the norovirus protease and polymerase proteins. This work investigated HuNV ProPol by determining the de novo polymerase activity, protein structure, and antiviral inhibition profile. The GII ProPol de novo enzymatic efficiencies (kcat/Km) for RNA templates and ribonucleotides were equal or superior to those of mature GII Pol on all templates measured. Furthermore, GII ProPol was the only enzyme form active on a poly(A) template. The first structure of the polymerase domain of HuNV ProPol in the unliganded state was determined by cryo-electron microscopy at a resolution of 2.6 Å. The active site and overall architecture of ProPol are similar to those of mature Pol. In addition, both galidesivir triphosphate and PPNDS inhibited polymerase activity of GII ProPol, with respective half-maximal inhibitory concentration (IC50) values of 247.5 µM and 3.8 µM. In both instances, the IC50 obtained with ProPol was greater than that of mature Pol, indicating that ProPol can exhibit different responses to antivirals. This study provides evidence that HuNV ProPol possesses overlapping and unique enzyme properties compared with mature Pol and will aid our understanding of the replication cycle of the virus.IMPORTANCEDespite human norovirus (HuNV) being a leading cause of acute gastroenteritis, the molecular mechanisms surrounding replication are not well understood. Reports have shown that HuNV replication generates precursor proteins from the viral polyprotein, one of which is the protease-polymerase (ProPol). This precursor is important for viral replication; however, the polymerase activity and structural differences between the precursor and mature forms of the polymerase remain to be determined. We show that substrate specificity and polymerase activity of ProPol overlap with, but is distinct from, the mature polymerase. We employ cryo-electron microscopy to resolve the first structure of the polymerase domain of ProPol. This shows a polymerase architecture similar to mature Pol, indicating that the interaction of the precursor with substrates likely defines its activity. We also show that ProPol responds differently to antivirals than mature polymerase. Altogether, these findings enhance our understanding of the function of the important norovirus ProPol precursor.

Automated detection and de novo structure modeling of nucleic acids from cryo-EM maps.

Cryo-electron microscopy (cryo-EM) is one of the most powerful experimental methods for macromolecular structure determination. However, accurate DNA/RNA structure modeling from cryo-EM maps is still challenging especially for protein-DNA/RNA or multi-chain DNA/RNA complexes. Here we propose a deep learning-based method for accurate de novo structure determination of DNA/RNA from cryo-EM maps at <5 Å resolutions, which is referred to as EM2NA. EM2NA is extensively evaluated on a diverse test set of 50 experimental maps at 2.0-5.0 Å resolutions, and compared with state-of-the-art methods including CryoREAD, ModelAngelo, and phenix.map_to_model. On average, EM2NA achieves a residue coverage of 83.15%, C4' RMSD of 1.06 Å, and sequence recall of 46.86%, which outperforms the existing methods. Moreover, EM2NA is applied to build the DNA/RNA structures with 10 to 5347 nt from an EMDB-wide data set of 263 unmodeled raw maps, demonstrating its ability in the blind model building of DNA/RNA from cryo-EM maps. EM2NA is fast and can normally build a DNA/RNA structure of <500 nt within 10 minutes.