Room-temperature Structure Research Articles

Joint X-ray/neutron structure of Lentinus similis AA9_A at room temperature.

Lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes which cleave polysaccharides oxidatively and are important in pathogen biology, carbon cycling and biotechnology. The Lentinus similis family AA9 isoform A (LsAA9_A) has been extensively studied as a model system because its activity towards smaller soluble saccharide substrates has allowed detailed structural characterization of its interaction with a variety of substrates by X-ray crystallography at high resolution. Here, the joint X-ray/neutron room-temperature crystallographic structure of carbohydrate-free LsAA9_A in the copper(II) resting state refined against X-ray and neutron data at 2.1 and 2.8 Å resolution, respectively, is presented. The results provide an experimental determination of the protonation states of the copper(II)-coordinating residues and second-shell residues in LsAA9_A, paving the way for future neutron crystallographic studies of LPMO-carbohydrate complexes.

Room-temperature serial synchrotron crystallography of the human phosphatase PTP1B.

Room-temperature X-ray crystallography provides unique insights into protein conformational heterogeneity, but obtaining sufficiently large protein crystals is a common hurdle. Serial synchrotron crystallography (SSX) helps to address this hurdle by allowing the use of many medium- to small-sized crystals. Here, a recently introduced serial sample-support chip system has been used to obtain the first SSX structure of a human phosphatase, specifically protein tyrosine phosphatase 1B (PTP1B) in the unliganded (apo) state. In previous apo room-temperature structures, the active site and allosteric sites adopted alternate conformations, including open and closed conformations of the active-site WPD loop and of a distal allosteric site. By contrast, in our SSX structure the active site is best fitted with a single conformation, but the distal allosteric site is best fitted with alternate conformations. This observation argues for additional nuance in interpreting the nature of allosteric coupling in this protein. Overall, our results illustrate the promise of serial methods for room-temperature crystallography, as well as future avant-garde crystallography experiments, for PTP1B and other proteins.

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme.

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.

Pink-Beam Serial Synchrotron Crystallography at Pohang Light Source II

Serial crystallography (SX) enables the determination of room-temperature structures with minimal radiation damage. The photon flux of the pink beam of 1.2% bandwidth (BW) is one order higher than that of the monochromatic beam from a silicon crystal monochromator, and the energy resolution of 1.2% BW is enough to solve the structure; therefore, it is useful to use the pink beam for time-resolved serial synchrotron crystallography (SSX). Here, we demonstrate a pink-beam serial synchrotron crystallographic study at the 1C beamline at the Pohang Light Source II. Lysozyme crystals embedded in a beef tallow injection matrix were delivered through a syringe into the X-ray interaction point. Pink-beam SSX was performed with different X-ray exposure positions to the injection stream (center and edge) and X-ray exposure times (50 and 100 ms). All lysozyme crystal structures were successfully determined at a high resolution of 1.7 Å. Background analysis showed that X-ray diffraction data exposed to the edge of the injection stream could improve the signal-to-noise ratio. All the diffraction data and room-temperature lysozyme structures were comprehensively compared. The data collection strategy and analysis will be helpful in further pink-beam SSX experiments and their applications.

Room-temperature structure, magnetic, and magnetocaloric properties of (La0.8-xNdx)Sr0.2MnO3(0 ≤ x ≤ 0.2)

In this study, the impacts of Nd doping on the structural, morphological, magnetic, and magnetocaloric properties of (La0.8-xNdx)Sr0.2MnO3 (0 ≤ x ≤ 0.2) (LNSMO) have been investigated. LNSMO has been synthesized using the sol-gel technique (SG). XRD analysis revealed that LNSMO has a rhombohedral structure and belongs to the R-3c space group (No. 167). In addition, the XRD refinement using Fullprof software demonstrated that the cell volume and lattice parameters gradually reduced as Nd doping increased, confirming the gradual replacement of the La sites by Nd. The fitting of the Mn 2p peak by X-ray photoelectron spectroscopy (XPS) confirmed that Mn is in mixed valence (Mn3+, Mn4+), which contributes to the double exchange interactions (DE). It is confirmed by M−H that the paramagnetic (PM) - ferromagnetic (FM) transition of LNSMO is a second-order magnetic phase transition. There is a significant change in the magnetocaloric effect (MCE) around room temperature. The results of the magnetization measurements indicate that a reduction in the Curie temperature (Tc) and an increase in the Nd doping cause an increase followed by a decrease in the maximum magnetic entropy change (-ΔSMmax). On the contrary, the relative cooling power (RCP) first decreases and then increases. Therefore, we propose La0.62Nd0.18Sr0.2MnO3 sample with Tc = 300 K, -ΔSMmax = 4.22 J/(kg·K) and RCP = 253.39 (J/kg) under an external magnetic field. This material is potentially one of the candidates that could be used to develop magnetic refrigeration technology.

Design of oxygen-doped TiZrHfNbTa refractory high entropy alloys with enhanced strength and ductility

Refractory high entropy alloys (RHEAs) are considered promising materials for high-temperature applications due to their thermal stability and high-temperature mechanical properties. However, most RHEAs have high density (>10 g/cm3) and exhibit limited ductility at low temperatures and softening at high temperatures. In this study, we show that oxygen-doping can be used as a new alloy design strategy for tailoring the mechanical behavior of the TiZrHfNbTa alloy: a novel low-density (7.98 g/cm3) ductile RHEA. Even though the material is a single-phase BCC with some oxides at room temperature, secondary BCC and HCP nano-lamellar structures start to form above 600 °C in addition to the nano-twins which are shown to be stable up to 1000 °C. This alloy shows superior strength and compressive ductility due to the nanoengineered microstructure. The present study sheds light on tailoring the strength-ductility balance in RHEAs by controlling the microstructure of novel RHEAs at the nanoscale via oxygen-doping.

Pbx(OH)y cluster formation and anomalous thermal behaviour in STI framework-type zeolites

For the first time, the structural investigation of a Pb-exchanged zeolite (Pb13.4(OH)10Al17.4Si54.6O144 ∙38H2O) with STI framework type, revealed a highly unusual and intriguing sudden volume increase under continuous heating. Understanding the fundamental mechanisms leading to such an unusual behaviour is essential for technological applications and interpretation of chemical bonding in zeolites. The dehydration was tracked in situ from 25 to 450 °C by single crystal X-ray diffraction, infrared and X-ray absorption spectroscopy. Further interpretation of the experimental observations was supported by ab initio molecular dynamics simulations. Initially, Pb-STI unit-cell volume contracts (ΔV = − 3.5%) from 25 to 100 °C. This agrees with the trend observed in STI zeolites. Surprisingly, at 125 °C, the framework expanded (ΔV = + 2%), adopting a configuration, which resembles that of the room temperature structure. Upon heating, the structure loses H2O but no de-hydroxylation occurred. The key mechanism leading to the sudden volume increase was found to be the formation of Pbx(OH)y clusters, which prevent the shrinking of the channels, rupture of the tetrahedral bonds and occlusion of the pores. This zeolite has therefore an increased thermal stability with respect to other STI metal-exchanged zeolites, with important consequences on its applications.

Structural transformation and thermodynamics of alloying CunAg55-n(n = 0–55) clusters on cooling from atomic simulations

Molecular dynamics simulations were performed to investigate the freezing process of CunAg55-n (n = 0–55) alloying cluster at the atomic scale. Potential energy and shape factors indicate the structural transformation accompanying with the atom motion. Pair-distribution function gives different atomic packing characteristics of some typical structures. Entropy combined with BP neural network is used to analyze the disorder degree of atomic packing in the binary alloying clusters as well as the influence of the number of Cu atoms on the entropy values. The simulation results show that the clusters having fewer or quantity Cu atoms prefer the perfect core–shell icosahedral structures at room temperature. Significant composition effects can be observed during cooling the small size alloying clusters. With changing compositions and decreasing the temperature, the packing structures change from disorder to order accompanying with the changes of atomic energy, shape factor, pair distribution functions, and entropy. In the restricted composition space having 55 atoms, the structure of the melt has an effect on the structural transformation during cooling and the room temperature structure.

Upgraded Combined Inject-and-Transfer System for Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) using an X-ray free-electron laser (XFEL) can be applied to determine the room-temperature structure of target molecules while minimizing radiation damage and visualizing molecular dynamics. In SFX, a sample delivery system is required to deliver microcrystals to the XFEL beam path in a serial manner. We recently developed a sample delivery method, the combined inject-and-transfer system (BITS), which is a hybrid method based on the injector and fixed-target scanning approach. In this study, we introduced recently upgraded hardware to move the injection needle in the direction of the XYZ-axis and a graphic user interface for user motion control. Furthermore, we report that the viscous solution containing 10% (w/v) PEG 3350 or PEG 6000 that is widely used for protein crystallization can be stably deposited on polyimide film with a hydrophobic surface without any special treatment. Moreover, the development of an inject-and-diffuse method for time-resolved studies with liquid applications in the BITS and its preliminary results are reported. This study provides up-to-date instrument information to SFX users using BITS and provides insights to instrument developers for SFX.

Room-temperature serial synchrotron crystallography of Drosophila cryptochrome.

Fixed-target serial crystallography allows the high-throughput collection of diffraction data from small crystals at room temperature. This methodology is particularly useful for difficult samples that have sensitivity to radiation damage or intolerance to cryoprotection measures; fixed-target methods also have the added benefit of low sample consumption. Here, this method is applied to the structure determination of the circadian photoreceptor cryptochrome (CRY), previous structures of which have been determined at cryogenic temperature. In determining the structure, several data-filtering strategies were tested for combining observations from the hundreds of crystals that contributed to the final data set. Removing data sets based on the average correlation coefficient among equivalent reflection intensities between a given data set and all others was most effective at improving the data quality and maintaining overall completeness. CRYs are light sensors that undergo conformational photoactivation. Comparisons between the cryogenic and room-temperature CRY structures reveal regions of enhanced mobility at room temperature in loops that have functional importance within the CRY family of proteins. The B factors of the room-temperature structure correlate well with those predicted from molecular-dynamics simulations.

Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature.

Room-temperature macromolecular crystallography allows protein structures tobe determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.

Suppressing lone-pair expression endows room-temperature cubic structure and high thermoelectric performance in GeTe-based materials

The stereochemical expression of ns2 lone-pair electrons in specific Ⅳ-Ⅵ group semiconductors is pivotal to lattice instability, which induces ferroelectric phase transition. In the thermoelectric device working temperature range, the distinct change of thermal expansion coefficient, however, is introduced by phase transition and then severely restrains material's critical applications. Herein, taking the example of thermoelectric GeTe, the hidden role of manipulating lone pair expression to control the crystal structure is theoretically unraveled. Moreover, it is experimentally demonstrated that LiBiTe2-alloyed GeTe possess a room-temperature cubic structure and competitive thermoelectric performance. Density functional theory calculations reveal that reducing the charge orientation caused by the stereochemical activity of cation lone-pair electrons in GeTe is an effective strategy to achieve room-temperature cubic structure. LiBiTe2 is found to be a novel alloying agent to realize crystal structure manipulating, carrier concentration optimization, multi-band convergence, and lattice softening. Benefiting from the optimized electrical and thermal transport properties, an ultrahigh ZTave of 1.22 within 300–773 K in cubic (GeTe)0.80(LiBiTe2)0.20 is achieved. This work provides a new insight into manipulating lone pair expression to control crystal structure, and promotes the development and application of cubic GeTe-based materials.

Combination of an inject-and-transfer system for serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) enables the determination of room-temperature crystal structures of macromolecules with minimized radiation damage and provides time-resolved molecular dynamics by pump-probe or mix-and-inject experiments. In SFX, a variety of sample delivery methods with unique advantages have been developed and applied. The combination of existing sample delivery methods can enable a new approach to SFX data collection that combines the advantages of the individual methods. This study introduces a combined inject-and-transfer system (BITS) method for sample delivery in SFX experiments: a hybrid injection and fixed-target scanning method. BITS allows for solution samples to be reliably deposited on ultraviolet ozone (UVO)-treated polyimide films, at a minimum flow rate of 0.5 nl min-1, in both vertical and horizontal scanning modes. To utilize BITS in SFX experiments, lysozyme crystal samples were embedded in a viscous lard medium and injected at flow rates of 50-100 nl min-1 through a syringe needle onto a UVO-treated polyimide film, which was mounted on a fixed-target scan stage. The crystal samples deposited on the film were raster scanned with an X-ray free electron laser using a motion stage in both horizontal and vertical directions. Using the BITS method, the room-temperature structure of lysozyme was successfully determined at a resolution of 2.1 Å, and thus BITS could be utilized in future SFX experiments.

Steering coherence in quantum dots by carriers injection via tunneling

Abstract Coherent control is a key experimental technique for quantum optics and quantum information processing. We demonstrate a new degree of freedom in coherent control of semiconductor quantum dot (QD) ensembles operating at room temperature using the tunneling injection (TI) processes in which charge carriers tunnel directly from a quantum well reservoir to QD confined states. The TI scheme was originally proposed and implemented to improve QD lasers and optical amplifiers, by providing a direct injection path of cold carriers thereby eliminating the hot carrier injection problem which enhances gain nonlinearity. The impact of the TI processes on the coherent time of the QDs was never considered, however. We show here that since the cold carriers that tunnel to the oscillating QD state are incoherent, the rate of injection determines the coherent time of the QDs thereby controlling coherent light–matter interactions. Coherent interactions by means of Rabi oscillations were demonstrated in absorption and for weak excitation pulses in the gain regime. However, Rabi oscillations are totally diminished under strong excitation pulses which increase the rate of stimulated emission, causing the tunneling processes to dominate what shortens the coherence time significantly. Since the tunneling rate, and hence, the coherence time, were controlled by the optical excitation and electrical bias, our finding paves the way for TI-based coherence switching on a sub-picosecond time scale in room-temperature semiconductor nanometric structures.

Study on Hydration and Dehydration of Ezetimibe by Terahertz Spectroscopy with Humidity-Controlled Measurements and Theoretical Analysis.

Understanding the solid-state transitions of active pharmaceutical ingredients (APIs) is essential for quality control since differences in their forms affect the bioavailability of APIs. Terahertz (THz) frequency-domain spectroscopy is suitable for such an application since it can sensitively probe the lattice phonon modes originating in the crystal structures. THz absorption spectra were obtained for ezetimibe (EZT) and ezetimibe monohydrate (EZT-MH), which have similar crystalline structures and belong to the same space group. The observed absorption spectrum of EZT matched well with the solid-state density functional theory (ss-DFT)-simulated spectrum for the structures at 0 K and room temperature (modeled using constrained unit cell volumes). However for EZT-MH, the ss-DFT spectrum of the room-temperature structure showed better correlation with the experimental THz spectrum than that of the simulated spectrum of the 0 K structures, suggesting that the EZT-MH crystal has greater anharmonic character. Gibbs free-energy curves were calculated, and EZT-MH was found to be more stable than pure EZT and water in a broad temperature range. The hydrate stability may be influenced by the existence of more hydrogen bonds in EZT-MH. The hydration and dehydration of EZT in a pure API tablet and formulation tablets were monitored using a THz spectrometer with a humidity-controlled sample chamber. The effect of the excipient in the formulation tablet on hydration was successfully confirmed by showing that the solid-state transition of the API with excipients is significantly slower than that without it. Under a relative humidity of 60%, hydration of EZT in a pure EZT tablet occurred in 200 min, while the hydration of EZT in a formulation tablet was 50 times slower.

The blue-green emission color of Ag+, Gd3+ co-activated CdWO4 phosphor via energy transfer for luminescence applications

Ag+, Gd+3-doped CdWO4 (co-doped CWO) phosphor nanopowders were synthesized by an eco-friendly method and then embedded into the polymer matrix to produce a flexible and transparent thin film. A novel phosphor for the excitation by an ultraviolet source, 980 nm diode laser, and proton radiation was reported at room temperature and crystal structure, luminescent properties, and energy transfer between Ag+ and Gd+3 ions were thoroughly investigated. Elemental composition and chemical state analyses were measured using X-ray photoelectron spectroscopy and energy dispersive X-ray spectroscopy studies. Crystal structure and phase identification were confirmed using powder X-ray diffraction measurement. Based on all results, the flexible phosphor film prepared has good thermal stability and efficient blue-green emission under UV light, laser, and proton excitation used in emissive displays applications.

Hit Expansion of a Noncovalent SARS-CoV-2 Main Protease Inhibitor

Inhibition of the SARS-CoV-2 main protease (Mpro) is a major focus of drug discovery efforts against COVID-19. Here we report a hit expansion of non-covalent inhibitors of Mpro. Starting from a recently discovered scaffold (The COVID Moonshot Consortium. Open Science Discovery of Oral Non-Covalent SARS-CoV-2 Main Protease Inhibitor Therapeutics. bioRxiv 2020.10.29.339317) represented by an isoquinoline series, we searched a database of over a billion compounds using a cheminformatics molecular fingerprinting approach. We identified and tested 48 compounds in enzyme inhibition assays, of which 21 exhibited inhibitory activity above 50% at 20 μM. Among these, four compounds with IC50 values around 1 μM were found. Interestingly, despite the large search space, the isoquinolone motif was conserved in each of these four strongest binders. Room-temperature X-ray structures of co-crystallized protein–inhibitor complexes were determined up to 1.9 Å resolution for two of these compounds as well as one of the stronger inhibitors in the original isoquinoline series, revealing essential interactions with the binding site and water molecules. Molecular dynamics simulations and quantum chemical calculations further elucidate the binding interactions as well as electrostatic effects on ligand binding. The results help explain the strength of this new non-covalent scaffold for Mpro inhibition and inform lead optimization efforts for this series, while demonstrating the effectiveness of a high-throughput computational approach to expanding a pharmacophore library.

Peierls distortion of the cobalt chain in the low-temperature structure of CoIn2

Abstract CoIn2 (Z. Metallkd. 1970, 61, 342–343) forms by reaction of the elements at 1470 K followed by annealing at 770 K for five days. The room temperature structure is orthorhombic (CuMg2 type, Fddd, a = 529.95(10), b = 940.49(13), c = 1785.8(3) pm, wR2 = 0.0563, 444 F 2 values, 17 variables) and shows a phase transition at 195(1) K (DSC data). The low-temperature modification crystallizes in the translationengleiche monoclinic subgroup C2/c and exhibits a new structure type (a = 933.7(7), b = 526.91(10), c = 1000.8(2) pm, β = 117.81(5)°, wR2 = 0.0374, 843 F 2 values, 30 variables). The structural phase transition is a consequence of a Peierls type distortion. The equidistant cobalt chains in HT-CoIn2 (270.1 pm, 175.2° Co–Co–Co) show pairwise dislocation in LT-CoIn2 with shorter (252.4 pm) and longer (284.1 pm) Co–Co distances. Each cobalt atom has coordination number 10 in the form of slightly distorted square antiprisms of indium, capped by cobalt on the rectangular faces. Density-of-states calculations reveal metallic behavior for both modifications. Integrated crystal orbital overlap populations featuring the bonding characteristics indicate a slightly higher intensity area for LT-CoIn2 along with a shift to lower energy, manifesting the stabilization by pair formation through Peierls distortion.

XFEL serial crystallography reveals the room temperature structure of methyl-coenzyme M reductase

Methyl-Coenzyme M Reductase (MCR) catalyzes the biosynthesis of methane in methanogenic archaea, using a catalytic Ni-centered Cofactor F430 in its active site. It also catalyzes the reverse reaction, that is, the anaerobic activation and oxidation, including the cleavage of the CH bond in methane. Because methanogenesis is the major source of methane on earth, understanding the reaction mechanism of this enzyme can have massive implications in global energy balances. While recent publications have proposed a radical-based catalytic mechanism as well as novel sulfonate-based binding modes of MCR for its native substrates, the structure of the active state of MCR, as well as a complete characterization of the reaction, remain elusive. Previous attempts to structurally characterize the active MCR-Ni(I) state have been unsuccessful due to oxidation of the redox- sensitive catalytic Ni center. Further, while many cryo structures of the inactive Ni(II)-enzyme in various substrates-bound forms have been published, no room temperature structures have been reported, and the structure and mechanism of MCR under physiologically relevant conditions is not known. In this study, we report the first room temperature structure of the MCRred1-silent Ni(II) form using an X-ray Free-Electron Laser (XFEL), with simultaneous X-ray Emission Spectroscopy (XES) and X-ray Diffraction (XRD) data collection. In celebration of the seminal contributions of inorganic chemist Dick Holm to our understanding of nickel-based catalysis, we are honored to announce our findings in this special issue dedicated to this remarkable pioneer of bioinorganic chemistry.

Nonstoichiometric effect on dielectric and large-signal electromechanical properties of environmentally friendly BNT-6BT ferroelectric ceramics

Although the nonstoichiometric influence on the small-signal dielectric and piezoelectric properties of (Bi0.5Na0.5)TiO3 (BNT)-based ferroelectrics has been studied extensively over the past decade, the features of large-signal electric field-induced strain (electrostrain), which are of particular importance to actuator devices, have not been thoroughly investigated. In this study, we used the solid-state reaction method to manufacture nonstoichiometric 0.94(Bi0.5+xNa0.5−x)TiO3-0.06BT (BNTx-6BT) ceramics, where x = 0.0–0.05, and investigated the nonstoichiometric effect on the dielectric and large-signal electromechanical properties, with special emphasis on the electrostrain properties. Our results suggest that the room-temperature phase structures of BNTx-6BT ceramics changed from a regular ferroelectric phase to a relaxor ferroelectric phase as the Bi/Na ratio increased from stoichiometric 50/50 to nonstoichiometric 55/45 owing to the nonstoichiometric effect on the long-range ferroelectric order. In the x = 0.02 nonstoichiometric composition, an ultrahigh and electrostrictive-type electrostrain of 0.53% was identified. Compared to their stoichiometric counterparts, nonstoichiometric compositions have stronger temperature stability during polarization, resulting in good temperature stability of the electrostrain. Our findings not only reveal the nonstoichiometric effect on the phase evolution and its impact on the dielectric and large-signal electromechanical properties of BNTx-6BT ceramics but also provide a new method for tailoring the large-signal electrostrain properties of BNT-based ceramics.