Negative Stain Electron Microscopy Research Articles

Nse5/6 is a negative regulator of the ATPase activity of the Smc5/6 complex.

The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein ‘holo-complex’ is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase activity, binding to the head-end of the complex to suppress turnover, irrespective of the DNA-bound status of the complex.

Network of Entamoeba histolytica HSP18.5 dimers formed by two overlapping [IV]-X-[IV] motifs.

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with low molecular weight that prevent the aggregation of proteins during stress conditions and maintain protein homeostasis in the cell. sHSPs exist in dynamic equilibrium as a mixture of oligomers of various sizes with a constant exchange of subunits between them. Many sHSPs form cage-like assemblies that may dissociate into smaller oligomers during stress conditions. We carried out the functional and structural characterization of a small heat shock protein, HSP18.5, from Entamoeba histolytica (EhHSP18.5). It showed a pH-dependent change in its oligomeric state, which varied from a tetramer to larger than 48-mer. EhHSP18.5 protected Nde I and lysozyme substrates from temperature and chemical stresses, respectively. The crystal structure of EhHSP18.5 was determined at a resolution of 3.28 Å in C2221 cell with four subunits in the asymmetric unit forming two non-metazoan sHSP-type dimers. Unlike the reported cage-like structures, EhHSP18.5 formed a network of linear chains of molecules in the crystal. Instead of a single [IV]-X-[IV] motif, EhHSP18.5 has two overlapping I/V-X-I/V sequences at the C-terminus giving rise to novel interactions between the dimers. Negative staining Electron Microscopy images of EhHSP18.5 showed the presence of multiple oligomers: closed structures of various sizes and long tube-like structures.

Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer.

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping.

Identification of a Dps contamination in Mitomycin-C–induced expression of Colicin Ia

Colicins are bacterial toxins targeting Gram-negative bacteria, including E. coli and related Enterobacteriaceae strains. Some colicins form ion-gated pores in the inner membrane of attacked bacteria that are lethal to their target. Colicin Ia was the first pore-forming E. coli toxin, for which a high-resolution structure of the monomeric full-length protein was determined. It is so far also the only colicin, for which a low-resolution structure of its membrane-inserted pore was reported by negative-stain electron microscopy. Resolving this structure at the atomic level would allow an understanding of the mechanism of toxin pore formation. Here, we report an observation that we made during an attempt to determine the Colicin Ia pore structure at atomic resolution. Colicin Ia was natively expressed by mitomycin-C induction under a native SOS promotor and purified following published protocols. The visual appearance in the electron microscope of negatively stained preparations and the lattice parameters of 2D crystals obtained from the material were highly similar to those reported earlier resulting from the same purification protocol. However, a higher-resolution structural analysis revealed that the protein is Dps (DNA-binding protein from starved cells), a dodecameric E. coli protein. This finding suggests that the previously reported low-resolution structure of a “Colicin Ia oligomeric pore” actually shows Dps.

Design of Alphavirus Virus-Like Particles Presenting Circumsporozoite Junctional Epitopes That Elicit Protection against Malaria.

The most advanced malaria vaccine, RTS,S, includes the central repeat and C-terminal domains of the Plasmodium falciparum circumsporozoite protein (PfCSP). We have recently isolated human antibodies that target the junctional region between the N-terminal and repeat domains that are not included in RTS,S. Due to the fact that these antibodies protect against malaria challenge in mice, their epitopes could be effective vaccine targets. Here, we developed immunogens displaying PfCSP junctional epitopes by genetic fusion to either the N-terminus or B domain loop of the E2 protein from chikungunya (CHIK) alphavirus and produced CHIK virus-like particles (CHIK-VLPs). The structural integrity of these junctional-epitope–CHIK-VLP immunogens was confirmed by negative-stain electron microscopy. Immunization of these CHIK-VLP immunogens reduced parasite liver load by up to 95% in a mouse model of malaria infection and elicited better protection than when displayed on keyhole limpet hemocyanin, a commonly used immunogenic carrier. Protection correlated with PfCSP serum titer. Of note, different junctional sequences elicited qualitatively different reactivities to overlapping PfCSP peptides. Overall, these results show that the junctional epitopes of PfCSP can induce protective responses when displayed on CHIK-VLP immunogens and provide a basis for the development of a next generation malaria vaccine to expand the breadth of anti-PfCSP immunity.

Structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate dehydrogenase complex (hKGDHc) revealed by cryo-EM and cross-linking mass spectrometry: Implications for the overall hKGDHc structure

BackgroundThe human mitochondrial alpha-ketoglutarate dehydrogenase complex (hKGDHc) converts KG to succinyl-CoA and NADH. Malfunction of and reactive oxygen species generation by the hKGDHc as well as its E1-E2 subcomplex are implicated in neurodegenerative disorders, ischemia-reperfusion injury, E3-deficiency and cancers. MethodsWe performed cryo-EM, cross-linking mass spectrometry (CL-MS) and molecular modeling analyses to determine the structure of the E2 component of the hKGDHc (hE2k); hE2k transfers a succinyl group to CoA and forms the structural core of hKGDHc. We also assessed the overall structure of the hKGDHc by negative-stain EM and modeling. ResultsWe report the 2.9 Å resolution cryo-EM structure of the hE2k component. The cryo-EM map comprises density for hE2k residues 151–386 - the entire (inner) core catalytic domain plus a few additional residues -, while residues 1–150 are not observed due to the inherent flexibility of the N-terminal region. The structure of the latter segment was also determined by CL-MS and homology modeling. Negative-stain EM on in vitro assembled hKGDHc and previous data were used to build a putative overall structural model of the hKGDHc. ConclusionsThe E2 core of the hKGDHc is composed of 24 hE2k chains organized in octahedral (8 × 3 type) assembly. Each lipoyl domain is oriented towards the core domain of an adjacent chain in the hE2k homotrimer. hE1k and hE3 are most likely tethered at the edges and faces, respectively, of the cubic hE2k assembly. General significanceThe revealed structural information will support the future pharmacologically targeting of the hKGDHc.

Ca2+-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum.

The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca2+ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca2+ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.

Amino Acid-Based Imidazole Ionic Liquid: A Novel Soft Matrix for Electrochemical Biosensing Applications

A new amino acid-based ionic liquid of 1-methyl-3-dodecane imidazolium aspartic acid ([Cₙmim]Asp IL, n = 4, 6, 8, 12, 14) was reported as a soft matrix to be used as a novel electrochemical glucose biosensor. [Cₙmim]Asp ILs were synthesized using a simple acid–base titration method and characterized by nuclear magnetic resonance spectroscopy, Fourier transform infrared (FT-IR) spectrum, scanning electron microscopy, negative-staining and cryogenic transmission electron microscopy, electrical conductivity, surface tension, dynamic light scattering, and UV–vis spectrum. [Cₙmim]Asp ILs possessed excellent conductivity and a large specific surface area, providing a biocompatible microenvironment to promote enzyme-catalyzed direct electron transfer while maintaining surface bioactivity and stability. It was found that the micelle structure could form in the [C₁₂mim]Asp IL solution when the concentration was beyond its critical micelle concentration. Glucose oxidase (GOx) could be uniformly dispersed and immobilized onto these micelles of the [C₁₂mim]Asp IL soft matrix through a hydrogen bond and physical absorption. Electrochemical experiments demonstrated that [C₁₂mim]Asp/GOx/Nafion/GCE achieved the best electrochemical performance toward glucose at a working potential of −0.33 V. The [C₁₂mim]Asp-based enzyme sensor displays a linear range response to glucose concentration from 0.001 to 0.58 and 0.58 to 12 mM and a low-end detection limit of 0.572 μM (S/N = 3). Additionally, the present glucose biosensor is robust to interfering molecules and shows acceptable reproducibility and practical application for real samples. The proposed amino acid-based ionic liquid can serve as a versatile and promising soft immobilization matrix for development of excellent biosensor applications.

Mechanisms of spacer acquisition by sequential assembly of the adaptation module in Synechocystis.

CRISPR–Cas immune systems process and integrate short fragments of DNA from new invaders as spacers into the host CRISPR locus to establish molecular memory of prior infection, which is also known as adaptation in the field. Some CRISPR–Cas systems rely on Cas1 and Cas2 to complete the adaptation process, which has been characterized in a few systems. In contrast, many other CRISPR–Cas systems require an additional factor of Cas4 for efficient adaptation, the mechanism of which remains less understood. Here we present biochemical reconstitution of the Synechocystis sp. PCC6803 type I-D adaptation system, X-ray crystal structures of Cas1–Cas2–prespacer complexes, and negative stained electron microscopy structure of the Cas4–Cas1 complex. Cas4 and Cas2 compete with each other to interact with Cas1. In the absence of prespacer, Cas4 but not Cas2 assembles with Cas1 into a very stable complex for processing the prespacer. Strikingly, the Cas1-prespacer complex develops a higher binding affinity toward Cas2 to form the Cas1–Cas2–prespacer ternary complex for integration. Together, we show a two-step sequential assembly mechanism for the type I-D adaptation module of Synechocystis, in which Cas4–Cas1 and Cas1–Cas2 function as two exclusive complexes for prespacer processing, capture, and integration.

GTP-dependent formation of straight tubulin oligomers leads to microtubule nucleation.

Nucleation of microtubules (MTs) is essential for cellular activities, but its mechanism is unknown because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. Here, combining rapid flush negative stain electron microscopy (EM) and kinetic analysis, we demonstrate that the formation of straight oligomers of critical size is essential for nucleation. Both GDP and GTP tubulin form single-stranded oligomers with a broad range of curvatures, but upon nucleation, the curvature distribution of GTP oligomers is shifted to produce a minor population of straight oligomers. With tubulin having the Y222F mutation in the β subunit, the proportion of straight oligomers increases and nucleation accelerates. Our results support a model in which GTP binding generates a minor population of straight oligomers compatible with lateral association and further growth to MTs. This study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.

Biophysical Studies of Interaction between Mycobacterial SepF and FtsZ

FtsZ is the bacterial tubulin homolog that forms Z-ring, which is a dynamic ring like structure, at the site of cell division to assemble the divisome and generate constrictive force for septum formation. SepF is the key protein that anchors the constricting Z-ring to the protruding septal membrane in Mycobacterium tuberculosis, via its interaction with the intrinsically disordered C-terminal tail of FtsZ. We characterized the cytosolic component of mycobacterial SepF, and its interaction with cognate FtsZ, using negative stained electron microscopy, small angle X-ray scattering, 90 degree angle light scattering and isothermal titration calorimetry.

Understanding how Mutations in Non-Muscle Myosin 2A Drive Human Disease

Non-muscle myosin 2A (NM2A) is a ubiquitously expressed myosin important for organizing actin filaments in areas of cell protrusion, cell adhesion, cell migration and cytokinesis and it is essential for early embryogenesis. Dephosphorylation of its regulatory light chain promotes formation of a compact inactive, ‘shutdown’ state in which the filament-forming tail is folded onto one of the two heads (known as the “10S” molecule). In this state, molecules cannot form filaments and the motors are inactivated. Over 80 mutations in the gene that encodes the heavy chain for NM2A (MYH9) have been described, most of which are single amino acid substitutions. Collectively these cause an autosomal-dominant disorder known as MYH9 disease, which result in a combination of symptoms from clotting disorders in which platelets are enlarged and bleeding time prolonged, aggregation of NM2A in neutrophils, cataracts, deafness and glomerulosclerosis. Here, using the cryo-electron microscopy structure we recently solved of shutdown smooth muscle myosin, we map known MYH9 mutations onto a homology model of NM2A. Multiple sequence alignment analysis reveals a high degree of conservation for residues involved in 10S-stabilising ionic interactions as well as many known mutation sites. Light meromyosin constructs (LMM is the region of the coiled-coil that assembles to form filaments) have been expressed, purified, and the effects of selected mutations have been assessed via negative stain electron microscopy. We suspect that these mutations may perturb the equilibrium between filamentous and 10S inactive molecules by actively destabilising the 10S conformation or by disrupting natural filament packing. With these results, we provide new structural insight on how mutations result in NM2A dysregulation and its implication in disease.

A FAB-Tag for Small Membrane Protein Structure Determination

The “resolution revolution” in electron microscopy left some proteins behind: small helical bundles that are almost entirely membrane embedded. Decorating a target with recombinant antibody fragments (Fab) that bind in a structurally specific and rigid manner can overcome limitations of small particle alignment, but antibody discovery campaigns are expensive, time-consuming, and are not guaranteed to work. Here we identify a helical epitope from the HIV envelope protein (gp41) that can be engineered as a contiguous extension of the first transmembrane helix of diverse membrane proteins. We demonstrate the utility of this maneuver by complexing a single Fab to three unrelated, small membrane proteins. The strategy permitted us to crystallize an otherwise structurally-intractable 30-kDa fluoride channel, as well as visualize Fab fragment decoration by negative stain EM. This 17-amino acid α-helical epitope can be bound by distinct, structurally characterized Fab fragments that can aid in structure determination of the target membrane protein.

GTP-Dependent Formation of Straight Oligomers Leads to Nucleation of Microtubules

Microtubule (MT) nucleation is essential for cellular activities, but its mechanism is not known because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. in cells, MTs are nucleated at tubulin concentrations significantly lower than those required for spontaneous nucleation in vitro. The high efficiency of nucleation is due to the synergistic effects of various cellular factors, but the underlying mechanism has not been clarified yet. Here, combining negative stain electron microscopy and kinetic analysis, we demonstrate that the formation of single-stranded straight oligomers with critical size is essential for nucleation in vitro.

Structure and Function of Abnormal Spindle-Like, Microtubule Associated Protein (ASPM)

Human autosomal recessive primary microcephaly (MCPH) is a hereditary disorder associated with a small brain and associated intellectual disability. Mutations in the gene for Abnormal Spindle-like, Microtubule Associated protein (ASPM) are the most common causes of MCPH. ASPM, a large (∼400kDa) protein, is important in mitosis. It anchors the mitotic spindle at the spindle poles and is essential for normal cytokinesis. However, the complete structure-function of ASPM is not well understood.The N-terminal domain of ASPM contains 2 tandem calponin homology (CH) domains and binds microtubules. However the location of the specific binding site is unclear. Two tandem CH domains typically bind actin, whereas single CH domains bind microtubules suggesting the CH domains of ASPM bind actin. The region following the CH domains contains 81 isoleucine-glutamine (IQ) motifs, known to bind light chains such as calmodulin. These motifs form a single alpha-helix to which the light chains bine, suggesting an overall elongated structure for ASPM.Using a combination of mass spectrometry and flow cell assays, we determined that the tandem CH domains of ASPM bind to microtubules, and not actin, suggesting that the CH domains may be the key binding site of ASPM for microtubules within the N-terminus. Negative stain electron microscopy of a central 21 IQ motif-containing domain revealed a long semi-flexible molecule, fully decorated with light chains. These data thus start to reveal the structure and function of ASPM.

Structural Studies of the Neuronal Apoptotic Complex of the Prongf-P75NTR-Sortilin

One of the prominent causes of neurodegenerative diseases is the dysregulation of the p75 neurotrophin receptor (p75NTR)-mediated signaling. Mature nerve growth factor (NGF) binds to p75NTR and the co-receptor tyrosine kinase a (TrkA) to maintain the neuronal proliferation, whereas the precursor of NGF (proNGF) interacts with p75NTR and sortilin, leading to neuronal apoptosis. The imbalance of the two signaling activities has strongly associated with Alzheimer's Disease and neurodegeneration. To elucidate the molecular mechanism of the proNGF-p75NTR-sortilin complex formation, we generated the extracellular domains of the target proteins and characterized the protein bindings in vitro. The extracellular domain complex will show the key structural motifs for protein-protein interactions, since the extracellular domains drive the complex formation. We used the Sf9 insect cell to perform heterologous overexpression, maintaining the simple post-glycosylation for membrane proteins. Previous study showed that sortilin forms a dimer in an acidic environment. However, negative-stain electron microscopy showed that the ternary complex of the proNGF-p75NTR-sortilin was formed in a dimeric assembly and was not sensitive to the environmental pH. This is likely that the binding interface may not be via the interaction between sortilins. To unravel the mechanistic view and identify the key residues in the binding interface, we are pursuing single-particle electron cryogenic microscopy (cryo-EM) to determine a high-resolution structure of the ternary complex. The result will provide a structural insight into the development of new medical interventions for treating neurodegenerative disorders.

Inference of molecular structure for characterization and improvement of clinical grade immunocytokines

The use of immunomodulatory agents for the treatment of cancer is gaining a growing biopharmaceutical interest. Antibody-cytokine fusion proteins, namely immunocytokines, represent a promising solution for the regulation of the immune system at the site of disease. The three-dimensional arrangement of these molecules can profoundly influence their biological activity and pharmacokinetic properties. Structural techniques might provide important insight in the 3D arrangement of immunocytokines. Here, we performed structure investigations on clinical grade fusion proteins L19-IL2, IL12-L19L19 and L19L19-IL2 to elucidate their quaternary organization. Crystallographic characterization of the common L19 antibody fragment at a resolution of 2.0-Å was combined with low-resolution studies of the full-length chimeric molecules using small-angle synchrotron X-ray scattering (SAXS) and negative stain electron microscopy. Characterization of the full-length quaternary structures of the immunocytokines in solution by SAXS consistently supported the diabody structure in the L19-IL2 immunocytokine and allowed generation of low-resolution models of the chimeric proteins L19L19-IL2 and IL12-L19L19. Comparison with 3D reconstructions obtained from negative-stain electron microscopy revealed marked flexibility associated to the linker regions connecting the cytokine and the antibody components of the chimeric proteins. Collectively, our results indicate that low-resolution molecular structure characterizations provide useful complementary insights for the quality control of immunocytokines, constituting a powerful tool to guide the design and the subsequent optimization steps towards clinical enhancement of these chimeric protein reagents.

Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

BackgroundDNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators.ResultsThis study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions.ConclusionKfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

Newcastle Disease Virus-Like Particles Displaying Prefusion-Stabilized SARS-CoV-2 Spikes Elicit Potent Neutralizing Responses.

The COVID-19 pandemic highlights an urgent need for vaccines that confer protection from SARS-CoV-2 infection. One approach to an effective COVID-19 vaccine may be through the display of SARS-CoV-2 spikes on the surface of virus-like particles, in a manner structurally mimicking spikes on a native virus. Here we report the development of Newcastle disease virus-like particles (NDVLPs) displaying the prefusion-stabilized SARS-CoV-2 spike ectodomain (S2P). Immunoassays with SARS-CoV-2-neutralizing antibodies revealed the antigenicity of S2P-NDVLP to be generally similar to that of soluble S2P, and negative-stain electron microscopy showed S2P on the NDVLP surface to be displayed with a morphology corresponding to its prefusion conformation. Mice immunized with S2P-NDVLP showed substantial neutralization titers (geometric mean ID50 = 386) two weeks after prime immunization, significantly higher than those elicited by a molar equivalent amount of soluble S2P (geometric mean ID50 = 17). Neutralizing titers at Week 5, two weeks after a boost immunization with S2P-NDVLP doses ranging from 2.0 to 250 μg, extended from 2125 to 4552, and these generally showed a higher ratio of neutralization versus ELISA than observed with soluble S2P. Overall, S2P-NDVLP appears to be a promising COVID-19 vaccine candidate capable of eliciting substantial neutralizing activity.

CopR, a Global Regulator of Transcription to Maintain Copper Homeostasis in Pyrococcus furiosus.

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analyzed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterize the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 μM copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the regulatory network that enables Pyrococcus to respond to increased copper concentrations.