Domain Rearrangements Research Articles

Influence of uniaxial tensile strain on magnetization behavior of Fe-9wt%Ni steel

This study investigated the effects of uniaxial tensile strain on the magnetization process of an Fe-9wt%Ni (9Ni) alloy to increase understanding of its magnetic behavior. As the applied strain was increased from 0 to 14 %, the magnetization curve changed remarkably, particularly under a high-intensity magnetic field, and magnetic flux density, relative permeability, remanence, and coercive force degraded significantly, especially at strains greater than the upper yield point. Increase in tensile strain caused increase in hysteresis losses, primarily because strain induced crystal defects, which affected the rearrangement of magnetic domains. Increased dislocation density was the primary contributor to the observed deterioration in magnetic properties; changes in texture and phase had little influence. This finding indicates that strain-induced deformation strongly affects the magnetic behavior of 9Ni alloys; this insight will be useful in research aimed at mitigating welding defects such as arc blow, that are attributed to the magnetization of Ni steel.

Structural Dynamics of E6AP E3 Ligase HECT Domain and Involvement of a Flexible Hinge Loop in the Ubiquitin Chain Synthesis Mechanism.

Ubiquitin (Ub) ligases E3 are important factors in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe and a C-lobe that are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for the HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of the HECT domain remain unclear. Here, we provide the first direct demonstration of the structural dynamics of the HECT domain using high-speed atomic force microscopy at the nanoscale. We also found that the flexibility of the hinge loop has a great impact not only on its structural dynamics but also on the formation mechanism of free Ub chains.

Regulation of the cell division hydrolase RipC by the FtsEX system in Mycobacterium tuberculosis

The FtsEX complex regulates, directly or via a protein mediator depending on bacterial genera, peptidoglycan degradation for cell division. In mycobacteria and Gram-positive bacteria, the FtsEX system directly activates peptidoglycan-hydrolases by a mechanism that remains unclear. Here we report our investigation of Mycobacterium tuberculosis FtsEX as a non-canonical regulator with high basal ATPase activity. The cryo-EM structures of the FtsEX system alone and in complex with RipC, as well as the ATP-activated state, unveil detailed information on the signal transduction mechanism, leading to the activation of RipC. Our findings indicate that RipC is recognized through a “Match and Fit” mechanism, resulting in an asymmetric rearrangement of the extracellular domains of FtsX and a unique inclined binding mode of RipC. This study provides insights into the molecular mechanisms of FtsEX and RipC regulation in the context of a critical human pathogen, guiding the design of drugs targeting peptidoglycan remodeling.

Structure of full-length cobalamin-dependent methionine synthase and cofactor loading captured in crystallo

Cobalamin-dependent methionine synthase (MS) is a key enzyme in methionine and folate one-carbon metabolism. MS is a large multi-domain protein capable of binding and activating three substrates: homocysteine, folate, and S-adenosylmethionine for methylation. Achieving three chemically distinct methylations necessitates significant domain rearrangements to facilitate substrate access to the cobalamin cofactor at the right time. The distinct conformations required for each reaction have eluded structural characterization as its inherently dynamic nature renders structural studies difficult. Here, we use a thermophilic MS homolog (tMS) as a functional MS model. Its exceptional stability enabled characterization of MS in the absence of cobalamin, marking the only studies of a cobalamin-binding protein in its apoenzyme state. More importantly, we report the high-resolution full-length MS structure, ending a multi-decade quest. We also capture cobalamin loading in crystallo, providing structural insights into holoenzyme formation. Our work paves the way for unraveling how MS orchestrates large-scale domain rearrangements crucial for achieving challenging chemistries.

Asymmetric gating of a human hetero-pentameric glycine receptor

Hetero-pentameric Cys-loop receptors constitute a major type of neurotransmitter receptors that enable signal transmission and processing in the nervous system. Despite intense investigations into their working mechanism and pharmaceutical potentials, how neurotransmitters activate these receptors remains unclear due to the lack of high-resolution structural information in the activated open state. Here we report near-atomic resolution structures resolved in digitonin consistent with all principle functional states of the human α1β GlyR, which is a major Cys-loop receptor that mediates inhibitory neurotransmission in the central nervous system of adults. Glycine binding induces cooperative and symmetric structural rearrangements in the neurotransmitter-binding extracellular domain but asymmetrical pore dilation in the transmembrane domain. Symmetric response in the extracellular domain is consistent with electrophysiological data showing cooperative glycine activation and contribution from both α1 and β subunits. A set of functionally essential but differentially charged amino acid residues in the transmembrane domain of the α1 and β subunits explains asymmetric activation. These findings provide a foundation for understanding how the gating of the Cys-loop receptor family members diverges to accommodate specific physiological environments.

Revealing Deformation Mechanisms in Polymer-Grafted Thermoplastic Elastomers via In Situ Small-Angle X-ray Scattering.

The tunable properties of thermoplastic elastomers (TPEs), through polymer chemistry manipulations, enable these technologically critical materials to be employed in a broad range of applications. The need to "dial-in" the mechanical properties and responses of TPEs generally requires the design and synthesis of new macromolecules. In these designs, TPEs with nonlinear macromolecular architectures outperform the mechanical properties of their linear copolymer counterparts, but the differences in the deformation mechanism providing enhanced performance are unknown. Here, in situ small-angle X-ray scattering (SAXS) measurements during uniaxial extension reveal distinct deformation mechanisms between a commercially available linear poly(styrene)-poly(butadiene)-poly(styrene) (SBS) triblock copolymer and the grafted SBS version containing grafted poly(styrene) (PS) chains from the poly(butadiene) (PBD) midblock. The neat SBS (φSBS = 100%) sample deforms congruently with the macroscopic dimensions, with the domain spacing between spheres increasing and decreasing along and transverse to the stretch direction, respectively. At high extensions, end segment pullout from the PS-rich domains is detected, which is indicated by a disordering of SBS. Conversely, the PS-grafted SBS that is 30 vol % SBS and 70% styrene (φSBS = 30%) exhibits a lamellar morphology, and in situ SAXS measurements reveal an unexpected deformation mechanism. During deformation, there are two simultaneous processes: significant lamellar domain rearrangement to preferentially orient the lamellae planes parallel to the stretch direction and crazing. The samples whiten at high strains as expected for crazing, which corresponds with the emergence of features in the 2D SAXS pattern during stretching consistent with fibril-like structures that bridge the voids in crazes. The significant domain rearrangement in the grafted copolymers is attributed to the new junctions formed across multiple PS domains by the grafting of a single chain. The in situ SAXS measurements provide insights into the enhanced mechanical properties of grafted copolymers that arise through improved physical cross-linking that leads to nanostructure domain reorientation for self-reinforcement and craze formation where fibrils help to strengthen the polymer.

Identification of hotspots in synthetic peptide inhibitors of the FOXO4:p53 interaction

Forkhead box protein O4 (FOXO4) plays a pivotal role in cellular senescence by binding to and inactivating p53. Consequently, misregulation of the FOXO4:p53 complex is associated with numerous diseases. Targeting the FOXO4-p53 interface has been achieved using a synthetic D-retro-inverso (DRI) peptide derived from the forkhead-homology domain of FOXO4 (FOXO4-FDH), also known as DRI (FOXO4-FHD residues 91–124). However, a comprehensive understanding of the key amino acids driving the interaction between DRI and p53 remains incomplete. While previous publications have demonstrated a robust interaction between the forkhead homology domain of FOXO4 (FOXO4-FHD) and the transactivation domain of p53 (p53-TAD), emerging evidence suggests that the interaction within the binary complex forms a highly interconnected network, including a predicted interaction between FOXO4-FHD and the DNA-binding domain of p53 (p53-DBD). In this study, we investigated the DRI: p53-DBD interaction by measuring the binding affinities of DRI and the native peptide of FOXO4, from which it is derived, to p53-DBD using microscale thermophoresis and computational modeling. Our in vitro measurements reveal that DRI binds to p53-DBD with high affinity (Kd ~50 nM), while the native peptide exhibits significantly weaker binding affinity (Kd ~2.5 mM), implying distinct modes of interaction. Subsequently, we created an in silico model of the DRI: p53-DBD interaction, which we analyzed to identify potential interaction hotspots. The analysis of this model suggests that a truncated DRI peptide (FOXO4-FHD amino acids 101–109) retains the majority of the binding affinity, as subsequently demonstrated in vitro (Kd ~40 nM). Collectively, this data furnishes molecular-level insights that contribute to the understanding of the interplay of the amino acids between DRI and p53, further supporting the notion of domain rearrangement or refolding during the formation of the FOXO4:p53 complex. In addition, this data provides an additional basis for the design of small molecules aimed at inhibiting this interaction.

Electrocaloric effect in pure and NaNbO3-modified Na0.5Bi0.5TiO3–BaTiO3 solid solutions near the morphotropic phase boundary

Morphotropic phase boundary of ferroelectric solid solutions attracts interest with respect to not only improved piezoelectric properties but also the electrocaloric effect, which is attractive to build new-generation cooling devices. In the present study, the electrocaloric effect in (1 − x − y)Na0.5Bi0.5TiO3–xBaTiO3–yNaNbO3 near its morphotropic phase boundary is studied by direct measurements of temperature change ΔT. ΔT maximum is observed in the region of depolarization temperature, where the major contribution comes from an entropy jump at the electric field-induced phase transition. Differences between ΔT values measured when an electric field pulse is applied and removed are explained by slow domain rearrangement in the ferroelectric phase and metastability of phases close to the depolarization temperature. Among studied compositions, the highest value of ΔT is obtained for the compositions 0.94Na0.5Bi0.5TiO3–0.06BaTiO3 and 0.93Na0.5Bi0.5TiO3–0.06BaTiO3–0.01NaNbO3 in the region of depolarization temperature. Examples of the comparison of the directly measured values of ΔT and the values calculated using the Maxwell relation revealing inconsistency between the two methods in the ferroelectric and the nonferroelectric phase are presented.

Anti-FVIII antibodies in Black and White hemophilia A subjects: do F8 haplotypes play a role?

The most common complication in hemophilia A (HA) treatment, affecting 25% to 30% of patients with severe HA, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8 haplotypes H1 to H5 are defined by nonsynonymous single-nucleotide polymorphisms encoding sequence variations at FVIII residues 1241, 2238, and 484. Haplotypes H2 to H5 are more prevalent in individuals with Black African ancestry, whereas 80% to 90% of the White population has the H1 haplotype. This study used an established multiplex fluorescence immunoassay to determine anti-FVIII antibody titers in plasma from 394 individuals with HA (188 Black, 206 White), measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3/H5, and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic assay and linear B-cell epitopes characterized using peptide microarrays. FVIII-reactive antibodies were readily detected in most individuals with HA, with higher titers in those with a current inhibitor, as expected. Neither total nor inhibitory antibody titers correlated with F8 haplotype mismatches, andpeptides with D1241E and M2238V polymorphisms did not comprise linear B-cell epitopes. Interestingly, compared with the full-length FVIII products, the BDD-FVIII proteins were markedly more reactive with plasma antibodies. The stronger immunoreactivity of BDD-FVIII suggests that B-domain removal might expose novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains.

Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status.

Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b' and a' domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b' and a' domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.

Tailoring Ni3ZnC0.7/graphite heterostructure in N-rich laminated porous carbon nanosheets for highly efficient microwave absorption

Bimetallic transition carbide nanoparticles (Ni3ZnC0.7) encapsulated with graphitic shells were successfully embedded into N-rich laminated porous carbon nanosheets (NC–ZnNi1.5) by one-step pyrolysis of bimetallic organic frameworks. It was found that C atoms penetrated octahedral interstitial spaces of the Ni lattice to form Ni3ZnC0.7. The charge states and distribution of metal atoms were influenced by the interstitial C atoms, which promoted polarization relaxation and facilitated dielectric loss. Simultaneously, the volatilization of Zn influenced recrystallization and rearrangement of the crystalline domains, facilitated graphitization and established a 3D conductive network to optimize the conductive loss. Besides, multi-level heterogeneous interface and nitrogen doping also further optimized the impedance matching. Benefiting from these advantages, the minimum reflection loss of NC-ZnNi1.5 was −69.1 dB at 12.7 GHz, and the effective absorption bandwidth was 6.5 GHz. The mechanism of bimetallic carbide's dielectric loss was explained in detail, providing a new pathway for the development of multi-component carbon-based microwave absorbers in the future.

Dissecting the Effect of Temperature on Hyperthermophilic Pf2001 Esterase Dimerization by Molecular Dynamics.

Pf2001 esterase (Pf2001) from Pyrococcus furiosus has hyperthermophilic properties and exerts a biocatalytic function in a dimeric state. Crystal structures revealed that the structural rearrangement of the cap domain is responsible for the Pf2001 dimer formation. However, the details of the cap domain remodeling and the effects of temperature on the dimerization process remain elusive at the molecular level, taking into account that experimental methods are difficult to capture the dynamic process of dimerization to some extent. Herein, four dimer models based on the monomeric crystal structure (PDB ID: 5G59) were constructed to investigate the conformational transition details and temperature effects in the dimerization by conventional molecular dynamics and accelerated molecular dynamics simulations. Our simulation results indicate that the monomer undergoes a conformational change into a "preparatory state" at high temperatures, which is more favorable for its transformation into a stable dimer. The subsequent free energy landscape analysis further identifies four intermediate states (from separated state to dimeric state) and discloses that a more accessible α-helix driven by stronger hydrophobic interactions induces a rearrangement of the cap domain, displaying a "tic-tac-toe" activation feature that is important for stabilizing the dimer interface and facilitating the formation of hydrophobic pockets. In addition, the electrostatic potential surface analysis illustrates that the weaker electrostatic repulsion (Lys and Arg) in the dimer interface at high temperatures is also a key factor for dimer stabilization. Altogether, our results can provide molecular-level insight into the dimer formation process of hyperthermophilic esterase and would be useful to understand the enzymatic specificity of α/β-hydrolase.

Conformational response to ligand binding of TMPRSS2, a protease involved in SARS-CoV-2 infection: Insights through computational modeling.

Thanks to the considerable research which has been undertaken in the last few years to improve our understanding of the biology and mechanism of action of SARS-CoV-2, we know how the virus uses its surface spike protein to infect host cells. The transmembrane prosthesis, serine 2 (TMPRSS2) protein, located on the surface of human cells, recognizes the cleavage site in the spike protein, leading to the release of the fusion peptide and entry of the virus into the host cells. Because of its role, TMPRSS2 has been proposed as a drug target to prevent infection by the virus. In this study, we aim to increase our understanding of TMPRSS2 using long scale microsecond atomistic molecular dynamics simulations, focusing on the conformational changes over time. The comparison between simulations conducted on the protein in the native (apo) and inhibited form (holo), has shown that in the holo form the inhibitor stabilizes the catalytic site and induces rearrangements in the extracellular domain of the protein. In turn, it leads to the formation of a new cavity in the vicinity of the ligand binding pocket that is stable in the microsecond time scale. Given the low specificity of known protease inhibitors, these findings suggest a new potential drug target site that can be used to improve TMPRSS2 specific recognition by newly designed inhibitors.

Agonist efficiency links binding and gating in a nicotinic receptor.

Receptors signal by switching between resting (C) and active (O) shapes ('gating') under the influence of agonists. The receptor's maximum response depends on the difference in agonist binding energy, O minus C. In nicotinic receptors, efficiency (η) represents the fraction of agonist binding energy applied to a local rearrangement (an induced fit) that initiates gating. In this receptor, free energy changes in gating and binding can be interchanged by the conversion factor η. Efficiencies estimated from concentration-response curves (23 agonists, 53 mutations) sort into five discrete classes (%): 0.56 (17), 0.51(32), 0.45(13), 0.41(26), and 0.31(12), implying that there are 5 C versus O binding site structural pairs. Within each class efficacy and affinity are corelated linearly, but multiple classes hide this relationship. η unites agonist binding with receptor gating and calibrates one link in a chain of coupled domain rearrangements that comprises the allosteric transition of the protein.

Crystal structures and solution conformations of HtrA from Helicobacter pylori reveal pH-dependent oligomeric conversion and conformational rearrangements

Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects over 50 % of the world's population, making it a major risk factor for chronic gastritis, ulcer diseases of the stomach and duodenum, MALT lymphoma, and gastric cancer. The clinical consequences of H. pylori infection are closely linked with the expression of virulence factors secreted by the bacterium. One such virulence factor is high temperature requirement A (HtrA), which possesses chaperone and serine protease activity. In the host stomach, HtrA secreted from H. pylori (HpHtrA) disrupts intercellular adhesions by cleaving epithelial adhesion proteins including E-cadherin and desmoglein-2. This disruption causes intercellular junctions to open, allowing the bacterium to pass through the epithelial barrier, access the intercellular space, and colonize the gastric mucosa. HtrA proteases are well known for their structural complexity, reflected in their diverse oligomer forms and multi-tasking activities in both prokaryotes and eukaryotes. In this study, we determined crystal structures and solution conformations of HpHtrA monomer and trimer, which revealed large domain rearrangements between them. Notably, this is the first report of a monomeric structure in the HtrA family. We further found a pH-dependent dynamic trimer-to-monomer conversion and concurrent conformational changes that seem closely linked with a pH-sensing ability through the protonation of certain Asp residues. These results advance our understanding of the functional roles and the related mechanisms of this protease in bacterial infection, which may shed light on the development of HtrA-targeted therapies for H. pylori-associated diseases.

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability.

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, including voltage-gated ion channels. This approach has been used primarily in heterologous expression systems to simultaneously measure membrane currents, the electrical manifestation of the channels' activity, and fluorescence measurements, reporting local domain rearrangements. Functional site-directed fluorometry combines electrophysiology, molecular biology, chemistry, and fluorescence into a single wide-ranging technique that permits the study of real-time structural rearrangements and function through fluorescence and electrophysiology, respectively. Typically, this approach requires an engineered voltage-gated membrane channel that contains a cysteine that can be tested by a thiol-reactive fluorescent dye. Until recently, the thiol-reactive chemistry used for the site-directed fluorescent labeling of proteins was carried out exclusively in Xenopus oocytes and cell lines, restricting the scope of the approach to primary non-excitable cells. This report describes the applicability of functional site-directed fluorometry in adult skeletal muscle cells to study the early steps of excitation-contraction coupling, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction. The present protocol describes the methodologies to design and transfect cysteine-engineered voltage-gated Ca2+ channels (CaV1.1) into muscle fibers of the flexor digitorum brevis of adult mice using in vivo electroporation and the subsequent steps required for functional site-directed fluorometry measurements. This approach can be adapted to study other ion channels and proteins. The use of functional site-directed fluorometry of mammalian muscle is particularly relevant to studying basic mechanisms of excitability.

Lactate: A critical regulator of cell proliferation via anaphase promoting complex remodeling

Lactate: A critical regulator of cell proliferation via anaphase promoting complex remodeling

Mechanistic aspects of tapasin-mediated antigen presentation revealed by structure of a tapasin/MHC-I complex

Abstract T cell immunity to viruses and cancers is crucially dependent on the cell surface display of major histocompatibility complex class I (MHC-I) molecules selectively loaded with high affinity peptides. Peptide loading occurs in the ER within a multimolecular assembly known as the peptide loading complex (PLC). A critical component of the PLC is tapasin, a 45 kDa membrane glycoprotein in whose absence the expression and stability of MHC-I are greatly reduced. To determine the mechanistic basis of tapasin-mediated peptide loading, we determined the crystal structures of human tapasin in complex with HLA-B44:05 as well as tapasin complexed to each of two well-characterized antibodies. The tapasin-stabilized peptide receptive state is characterized by distortions of the peptide binding groove, destabilization of the b 2-microglobulin interaction, and rearrangements of the membrane proximal Ig domains, all of which are reversed on high affinity peptide binding. Additionally, the structural footprints of the anti-tapasin antibodies confirm previous functional assays and together with the tapasin/MHC-I complex and mutagenesis data reveal dynamic aspects of tapasin function. Supported by the Intramural Research Program, NIAID, NIH.

Role of RNA Domain Structure and Orientation in the Coxsackievirus B3 Virulence Phenotype.

Coxsackievirus B3 (CVB3) is an enterovirus that causes diseases such as pancreatitis and myocarditis in humans. Approximately 10% of the CVB3 RNA genome consists of a highly structured 5' untranslated region (5' UTR) that is organized into six domains and contains a type I internal ribosome entry site (IRES). These features are common to all enteroviruses. Each RNA domain plays a vital role in translation and replication during the viral multiplication cycle. We used SHAPE-MaP chemistry to generate secondary structures of the 5' UTR from the avirulent strain CVB3/GA and the virulent strain CVB3/28. Our comparative models show how key nucleotide substitutions cause major restructuring of domains II and III of the 5' UTR in CVB3/GA. Despite these structural shifts, the molecule maintains several well-characterized RNA elements, which allows persistence of the unique avirulent strain. The results shed light on the 5' UTR regions serving as virulence determinants and those required for fundamental viral mechanisms. We used the SHAPE-MaP data to produce theoretical tertiary models using 3dRNA v2.0. These models suggest a compact conformation of the 5' UTR from the virulent strain CVB3/28 that brings critical domains into close contact. In contrast, the model of the 5' UTR from the avirulent strain CVB3/GA suggests a more extended conformation where the same critical domains are more separated. Our results suggest that the structure and orientation of RNA domains in the 5' UTR are responsible for low-efficiency translation, low viral titers, and absence of virulence observed during infection by CVB3/GA. IMPORTANCE Human enteroviruses, which include five different species and over 100 serotypes, are responsible for diseases ranging from mild respiratory infections to serious infections of pancreas, heart, and neural tissue. All enteroviral RNA genomes have a long and highly structured 5' untranslated region (5' UTR) containing an internal ribosome entry site (IRES). Major virulence determinants are located in the 5' UTR. We present RNA structure models that directly compare the 5' UTR derived from virulent and avirulent strains of the enterovirus coxsackievirus B3 (CVB3). The secondary-structure models show rearrangement of RNA domains known to be virulence determinants and conservation of structure in RNA elements known to be vital for translation and replication in the avirulent strain CVB3/GA. The tertiary-structure models reveal reorientation of RNA domains in CVB3/GA. Identifying the details of structure in these critical RNA domains will help direct antiviral approaches to this major human pathogen.

Structures of human primosome elongation complexes.

The synthesis of RNA-DNA primer by primosome requires coordination between primase and DNA polymerase α subunits, which is accompanied by unknown architectural rearrangements of multiple domains. Using cryogenic electron microscopy, we solved a 3.6 Å human primosome structure caught at an early stage of RNA primer elongation with deoxynucleotides. The structure confirms a long-standing role of primase large subunit and reveals new insights into how primosome is limited to synthesizing short RNA-DNA primers.