Structural Neutron Research Articles

XFEL structure of carbonic anhydrase II: a comparative study of XFEL, NMR, X-ray and neutron structures.

The combination of X-ray free-electron lasers (XFELs) with serial femtosecond crystallography represents cutting-edge technology in structural biology, allowing the study of enzyme reactions and dynamics in real time through the generation of `molecular movies'. This technology combines short and precise high-energy X-ray exposure to a stream of protein microcrystals. Here, the XFEL structure of carbonic anhydrase II, a ubiquitous enzyme responsible for the interconversion of CO2 and bicarbonate, is reported, and is compared with previously reported NMR and synchrotron X-ray and neutron single-crystal structures.

The solution structure of the heavy chain–only C5-Fc nanobody reveals exposed variable regions that are optimal for COVID-19 antigen interactions

Heavy chain-only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities than conventional antibodies. To address the challenge of SARS-CoV-2 coronavirus, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 virus neutralization potency. The fusion protein C5-Fc comprises two C5 domains attached to a glycosylated Fc region of a human IgG1 antibody and shows therapeutic efficacy invivo. Here, we have characterized the solution arrangement of the molecule. Two 1443DaN-linked glycans seen in the mass spectra of C5-Fc were removed and the glycosylated and deglycosylated structures were evaluated. Reduction of C5-Fc with 2-mercaptoethylamine indicated three interchain Cys-Cys disulfide bridges within the hinge. The X-ray and neutron Guinier RG values, which provide information about structural elongation, were similar at 4.1 to 4.2nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modeling based on Monte Carlo simulations resulted in 72,737 and 56,749 physically realistic trial X-ray and neutron structures, respectively. From these, the top 100 best-fit X-ray and neutron models were identified as representative asymmetric solution structures, similar to that of human IgG1, with good R-factors below 2.00%. Both C5 domains were solvent exposed, consistent with the functional effectiveness of C5-Fc. Greater disorder occurred in the Fc region after deglycosylation. Our results clarify the importance of variable and exposed C5 conformations in the therapeutic function of C5-Fc, while the glycans in the Fc region are key for conformational stability in C5-Fc.

ZA models of neutron and proton in scale electric field originated by the Planck particle

<p>A new ZA matter model has been established to explain why mass has the instinct property of electricity. It is shown that the pure energy particle to originate matter is an electric pulse called ZA0 and it was proved being the Planck particle. Matter formation was derived by theoretical analysis based on a suggested binary growth law of quantum mechanics. It is found that there is a smallest matter particle called as ZA1 with a same format of ZA0 but different size to form any other matter particles. Structures of electron, neutron and proton have been suggested and prediction of their mass meets experiments wonderfully. It was found that matter mass consists of two parts: one (a scale electric field) is electric pure mass coming from motion energy of the photon in minimum size and the other (the overlapped gravity field) coming from potential energy of the bent space. Sizes of basic matter particles were discussed based on the particle nature of isolated electric field. The size predictions can explain the weird experimental phenomenon that size of an electron is significantly larger than that of a neutron. It was found that electric charges in micro scale like γ-ray are represented at the zero potential nodes by opposite two momentum directions. It is shown by photoelectric effect experiments that electric force in micro by ZA model in form of light can be exchanged into the macro electric force to drive electrons in macro current. The proton ZA model can be applied to explain why many protons can be collaborated in a nucleus as the reactive binding force between protons and neutrons can overcome the electric repelling force between positive charges.</p>

Hirshfeld Atom Refinement of Metal-Organic Complexes: Treatment of Hydrogen Atoms Bonded to Transition Metals.

Hydrogen positions in hydrides play a key role in hydrogen storage materials and high-temperature superconductors. Our recently published study of five crystal structures of transition-metal-bound hydride complexes showed that using aspherical atomic scattering factors for Hirshfeld atom refinement (HAR) resulted in a systematic elongation of metal-hydrogen bonds compared to using spherical scattering factors with the Independent Atom Model (IAM). Even though only standard-resolution X-ray data was used, for the highest-quality data, we obtained excellent agreement between the X-ray and the neutron-derived bond lengths. We present an extended version of this study including 10 crystal structures of metal-organic complexes containing hydrogen atoms bonded to transition-metal atoms for which both X-ray and neutron data are available. The neutron structures were used as a benchmark, and the X-ray structures were refined by applying Hirshfeld atom refinement using various basis sets and DFT functionals in order to investigate the influence of the technical aspects on the length of metal-hydrogen bonds. The result of including relativistic effects in the Hamiltonian and using a cluster of multipoles simulating interactions with a crystal environment during wave function calculations was examined. The effect of the data quality on the final result was also evaluated. The study confirms that a high quality of experimental data is the key factor allowing us to obtain significant improvement in transition metal (TM)-hydrogen bond lengths from HAR in comparison with the IAM. Individual adjustments and better choices of the basis set can improve hydrogen positions. Average differences between TM-H bond lengths obtained with various DFT functionals upon including relativistic effects or between double-ζ and triple-ζ basis sets were not statistically significant. However, if all bonds formed by H atoms were considered, significant differences caused by different refinement strategies were observed. Finally, we examined the refinement of atomic thermal motions. Anisotropic refinement of hydrogen thermal motions with HAR was feasible only in some cases, and isotropically refined hydrogen thermal motions were in similar agreement with neutron values whether obtained with HAR or with the IAM.

New insights into the oxidation process from neutron and X-ray crystal structures of an O2-sensitive [NiFe]-hydrogenase.

[NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an O2-sensitive enzyme that is inactivated in the presence of O2 but the oxidized enzyme can recover its catalytic activity by reacting with H2 under anaerobic conditions. Here, we report the first neutron structure of [NiFe]-hydrogenase in its oxidized state, determined at a resolution of 2.20 Å. This resolution allowed us to reinvestigate the structure of the oxidized active site and to observe the positions of protons in several short hydrogen bonds. X-ray anomalous scattering data revealed that a part of the Ni ion is dissociated from the active site Ni-Fe complex and forms a new square-planar Ni complex, accompanied by rearrangement of the coordinated thiolate ligands. One of the thiolate Sγ atoms is oxidized to a sulfenate anion but remains attached to the Ni ion, which was evaluated by quantum chemical calculations. These results suggest that the square-planar complex can be generated by the attack of reactive oxygen species derived from O2, as distinct from one-electron oxidation leading to a conventional oxidized form of the Ni-Fe complex. Another major finding of this neutron structure analysis is that the Cys17S thiolate Sγ atom coordinating to the proximal Fe-S cluster forms an unusual hydrogen bond with the main-chain amide N atom of Gly19S with a distance of 3.25 Å, where the amide proton appears to be delocalized between the donor and acceptor atoms. This observation provides insight into the contribution of the coordinated thiolate ligands to the redox reaction of the Fe-S cluster.

Neutron crystallography and quantum chemical analysis of bilin reductase PcyA mutants reveal substrate and catalytic residue protonation states

PcyA, a ferredoxin-dependent bilin pigment reductase, catalyzes the site-specific reduction of the two vinyl groups of biliverdin (BV), producing phycocyanobilin. Previous neutron crystallography detected both the neutral BV and its protonated form (BVH+) in the wildtype (WT) PcyA-BV complex, and a nearby catalytic residue Asp105 was found to have two conformations (protonated and deprotonated). Semiempirical calculations have suggested that the protonation states of BV are reflected in the absorption spectrum of the WT PcyA-BV complex. In the previously determined absorption spectra of the PcyA D105N and I86D mutants, complexed with BV, a peak at 730nm, observed in the WT, disappeared and increased, respectively. Here, we performed neutron crystallography and quantum chemical analysis of the D105N-BV and I86D-BV complexes to determine the protonation states of BV and the surrounding residues and study the correlation between the absorption spectra and protonation states around BV. Neutron structures elucidated that BV in the D105N mutant is in a neutral state, whereas that in the I86D mutant is dominantly in a protonated state. Glu76 and His88 showed different hydrogen bonding with surrounding residues compared with WT PcyA, further explaining why D105N and I86D have much lower activities for phycocyanobilin synthesis than the WT PcyA. Our quantum mechanics/molecular mechanics calculations of the absorption spectra showed that the spectral change in D105N arises from Glu76 deprotonation, consistent with the neutron structure. Collectively, our findings reveal more mechanistic details of bilin pigment biosynthesis.

Analysis of discrepancies between D1S and R2S results of 2016 DD JET Campaign

In the frame of the WPJET3-DT Technology project within the EUROfusion Consortium program, neutronics experiments are being carried out in preparation for the current deuterium–tritium campaign on JET (DTE2). The experiments are conducted to validate the neutronics codes and tools used in ITER, thus reducing the related uncertainties and the associated risks in the machine operation. Recently, the shutdown dose rate (SDR) results of the 2016 DD campaign have been analyzed using the D1S and R2S computational methodologies showing relevant differences between the results provided by both methods. The greatest discrepancies, found in the SDR calculated in the second octant at 6 h after shutdown, are a factor 2 of difference in the total decay photon flux and a different peak location close to 0.511 MeV. This article presents a detailed analysis of the D1S and R2S simulations performed to find the causes of these discrepancies. The conclusions of the analysis determine that the use of the typical neutron and gamma energy structures used in common R2S simulations can tend to a photon flux significant overestimation or a shift in the output spectrum, respectively. In addition to alerting about this important problem in R2S calculations, this article also proposes a solution to mitigate each effect that can be implemented in R2S codes. The application of the proposed solutions significantly reduces the differences between the photon spectrum calculated with D1S and R2S methods.

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.

Constraining scalar-tensor theories using neutron star mass and radius measurements

We use neutron star mass and radius measurements to constrain the spontaneous scalarization phenomenon in scalar-tensor theories using Bayesian analysis. Neutron star structures in this scenario can be significantly different from the case of general relativity, which can be used to constrain the theory parameters. We utilize this idea to obtain lower bounds on the coupling parameter $\beta$ for the case of massless scalars. These constraints are currently weaker than the ones coming from binary observations, and they have relatively low precision due to the approximations in our method. Nevertheless, our results clearly demonstrate the power of the mass-radius data in testing gravity, and can be further improved with future observations. The picture is different for massive scalars, for which the same data is considerably less effective in constraining the theory parameters in an unexpected manner. We identify the main reason for this to be a large high-likelihood region in the parameter space where deviations from general relativity are relatively small. We hope this initial study to be an invitation to use neutron star structure measurements more commonly to test alternative theories in general.

The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and x-ray diffraction at room temperature.

The nonstructural protein 3 (NSP3) macrodomain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Mac1) removes adenosine diphosphate (ADP) ribosylation posttranslational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the coronavirus disease 2019 pandemic. Here, we determined neutron and x-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose–bound states. We characterize extensive solvation in the Mac1 active site and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase the potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a reevaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

Isospin asymmetry and quantum statistical interpretation of nucleons

In this paper, a statistical model introducing analytical expressions of [Formula: see text] dependent quantum statistical parton distributions is presented to explore proton and neutron structures. A merely theoretical maximum entropy method is applied to obtain thermodynamical variables determining parton distributions accurately. This study provides considering proton and neutron isospin asymmetry in order to improve the consistency of the framework achievements with the experimental results. The presented approach is extended to evaluate modified neutron structure in an [Formula: see text]-[Formula: see text] state. Also, the uniqueness of gluon distribution in proton and neutron is surveyed for both cases of lonely nucleon and [Formula: see text]-[Formula: see text] system. Eventually, the predicted unpolarized structure functions and the spin structure functions, only based on the maximum entropy method, are compared to CERN, HERMES, SLAC and the other data sets. Finally, the correlation of the obtained distributions with the experimental observations confirms the proposed statistical model.

New Atom Configuration Based on Proton and Electron as Electric Monopole Pairs and Exploration of Quarks and Higgs Particle

A new atom configuration was developed, based on proton and electron as electric monopole pairs deducted from “the theory of spin vector in motion” developed by us recently, and symmetry of group theory. New proton and neutron structures were proposed based on quarks’ mass and spin properties. According to proton and neutron’s configuration proposed, we concluded that the radioactivity of nucleus shall result directly from the electric repulsive force induced by symmetry breaking resulted from extra neutron(s) of an isotope. Based on inner structures of proton and neutron, we concluded that there is no “strong” force required to bond quarks in proton and neutron, the fused proton or neutron’s empty carriers or containers is the Higgs particle.

A protocol for production of perdeuterated OmpF porin for neutron crystallography

Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope 1H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope 2H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted by dissolving the protein in heavy water, complete deuterium labelling – perdeuteration – requires the protein to be expressed in heavy water with a deuterated carbon source. In this work, we developed an optimized method for large scale production of deuterium-labelled bacterial outer membrane protein F (OmpF) for NMX. OmpF was produced using deuterated media with different carbon sources. Mass spectrometry verified the integrity and level of deuteration of purified OmpF. Perdeuterated OmpF crystals diffracted X-rays to a resolution of 1.9 Å. This work lays the foundation for structural studies of membrane protein by neutron diffraction in future.

Neutron structures of Leishmania mexicana triosephosphate isomerase in complex with reaction-intermediate mimics shed light on the proton-shuttling steps.

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis that catalyses the interconversion of glyceraldehyde 3-phosphate and dihydroxy-acetone phosphate. This simple reaction involves the shuttling of protons mediated by protolysable side chains. The catalytic power of TIM is thought to stem from its ability to facilitate the deprotonation of a carbon next to a carbonyl group to generate an enediolate intermediate. The enediolate intermediate is believed to be mimicked by the inhibitor 2-phosphoglycolate (PGA) and the subsequent enediol intermediate by phosphoglycolohydroxamate (PGH). Here, neutron structures of Leishmania mexicana TIM have been determined with both inhibitors, and joint neutron/X-ray refinement followed by quantum refinement has been performed. The structures show that in the PGA complex the postulated general base Glu167 is protonated, while in the PGH complex it remains deprotonated. The deuteron is clearly localized on Glu167 in the PGA-TIM structure, suggesting an asymmetric hydrogen bond instead of a low-barrier hydrogen bond. The full picture of the active-site protonation states allowed an investigation of the reaction mechanism using density-functional theory calculations.

Exploring the halo phenomena of medium-mass nuclei having approximately Z= 40 with point-coupled parameters in complex momentum representations * *Supported by the National Natural Science Foundation of China (11875070) and the Natural Science Foundation of Anhui Province (1908085MA16)

To explore the properties of neutron-rich nuclei with approximately 40 protons, the density-dependent point coupling (DD-PC1) effective interaction parameter is adopted in the relativistic mean-field theory with the complex momentum representation (RMF-CMR). The calculated two-neutron separation energy (S 2n ) and root-mean-square (rms) radii support the halo structure that appear in Mo and Ru isotopic chains. Besides, the neutron skin structures appear in Kr and Sr isotopes. The conclusions drawn are also supported by the single-particle energy levels and their occupancy probability and density distribution. Particularly, the energy levels, which reduce to bound states or are approximately 0 MeV with a small orbital angular momentum, are suggested to provide the primary contribution to increasing the neutron radius. Moreover, the single-particle energy levels significantly reflect the shell structure. In addition, the neutron drip line nuclei for Kr, Sr, Mo, and Ru elements are proposed via the changes in S 2n .

Capturing the Catalytic Proton of Dihydrofolate Reductase: Implications for General Acid-Base Catalysis.

Acid–base catalysis, which involves one or more proton transfer reactions, is a chemical mechanism commonly employed by many enzymes. The molecular basis for catalysis is often derived from structures determined at the optimal pH for enzyme activity. However, direct observation of protons from experimental structures is quite difficult; thus, a complete mechanistic description for most enzymes remains lacking. Dihydrofolate reductase (DHFR) exemplifies general acid–base catalysis, requiring hydride transfer and protonation of its substrate, DHF, to form the product, tetrahydrofolate (THF). Previous X-ray and neutron crystal structures coupled with theoretical calculations have proposed that solvent mediates the protonation step. However, visualization of a proton transfer has been elusive. Based on a 2.1 Å resolution neutron structure of a pseudo-Michaelis complex of E. coli DHFR determined at acidic pH, we report the direct observation of the catalytic proton and its parent solvent molecule. Comparison of X-ray and neutron structures elucidated at acidic and neutral pH reveals dampened dynamics at acidic pH, even for the regulatory Met20 loop. Guided by the structures and calculations, we propose a mechanism where dynamics are crucial for solvent entry and protonation of substrate. This mechanism invokes the release of a sole proton from a hydronium (H3O+) ion, its pathway through a narrow channel that sterically hinders the passage of water, and the ultimate protonation of DHF at the N5 atom.

Visualization of hydrogen atoms in a perdeuterated lectin-fucose complex reveals key details of protein-carbohydrate interactions

Carbohydrate-binding proteins from pathogenic bacteria and fungi have been shown to be implicated in various pathological processes, where they interact with glycans present on the surface of the host cells. These interactions are part of the initial processes of infection of the host and are very important to study at the atomic level. Here, we report the room temperature neutron structures of PLL lectin from Photorhabdus laumondii in its apo form and in complex with deuterated L-fucose, which is, to our knowledge, the first neutron structure of a carbohydrate-binding protein in complex with a fully deuterated carbohydrate ligand. A detailed structural analysis of the lectin-carbohydrate interactions provides information on the hydrogen bond network, the role of water molecules, and the extent of the CH-π stacking interactions between fucose and the aromatic amino acids in the binding site.

Visualization of H atoms in the X-ray crystal structure of photoactive yellow protein: Does it contain low-barrier hydrogen bonds?

The hydrogen bond (HB) between 4-hydroxycinnamic acid (HC4) and glutamic acid E46 of photoactive yellow protein is exceptionally strong. In the 0.82-å resolution X-ray structure for this protein (PDB ID: 1NWZ), the OH…O distance is only 2.57 å. The position of the H atom between these two O atoms has not been determined in that structure, and in the absence of that information, it is impossible to determine whether or not this HB is a low-barrier HB (LBHB), as was proposed recently based on neutron structures of this protein (Yamaguchi et al., Proceedings of the National Academy of Sciences of the United States of America, 2009, 106: 440-444). Residual electron density maps computed using the 1NWZ data reveal that this H atom is 0.92 å from the Oε2 atom of E46 and 1.67 å from the O4 ' of HC4, and that the OH…O bond angle is 167°. These observations indicate that E46 is protonated, and HC4 is deprotonated, as was originally suggested, and that the HB in question is not an LBHB.

Neutron and X-ray crystal structures of Lactobacillus brevis alcohol dehydrogenase reveal new insights into hydrogen-bonding pathways.

Lactobacillus brevis alcohol dehydrogenase (LbADH) is a well studied homotetrameric enzyme which catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. LbADH is stable and enzymatically active at elevated temperatures and accepts a broad range of substrates, making it a valuable tool in industrial biocatalysis. Here, the expression, purification and crystallization of LbADH to generate large, single crystals with a volume of up to 1 mm3 suitable for neutron diffraction studies are described. Neutron diffraction data were collected from an H/D-exchanged LbADH crystal using the BIODIFF instrument at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany to a resolution dmin of 2.15 Å in 16 days. This allowed the first neutron crystal structure of LbADH to be determined. The neutron structure revealed new details of the hydrogen-bonding network originating from the ion-binding site of LbADH and provided new insights into the reasons why divalent magnesium (Mg2+) or manganese (Mn2+) ions are necessary for its activity. X-ray diffraction data were obtained from the same crystal at the European Synchrotron Radiation Facility (ESRF), Grenoble, France to a resolution dmin of 1.48 Å. The high-resolution X-ray structure suggested partial occupancy of Mn2+ and Mg2+ at the ion-binding site. This is supported by the different binding affinity of Mn2+ and Mg2+ to the tetrameric structure calculated via free-energy molecular-dynamics simulations.

The Neutron Macromolecular Crystallography Instruments at Oak Ridge National Laboratory: Advances, Challenges, and Opportunities

The IMAGINE and MaNDi instruments, located at Oak Ridge National Laboratory High Flux Isotope Reactor and Spallation Neutron Source, respectively, are powerful tools for determining the positions of hydrogen atoms in biological macromolecules and their ligands, orienting water molecules, and for differentiating chemical states in macromolecular structures. The possibility to model hydrogen and deuterium atoms in neutron structures arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction studies at the 1.5–2.5 Å resolutions, which are typical for protein crystals. Neutrons have the additional benefit for structural biology of not inducing radiation damage to protein crystals, which can be critical in the study of metalloproteins. Here we review the specifications of the IMAGINE and MaNDi beamlines and illustrate their complementarity. IMAGINE is suitable for crystals with unit cell edges up to 150 Å using a quasi-Laue technique, whereas MaNDi provides neutron crystallography resources for large unit cell samples with unit cell edges up to 300 Å using the time of flight (TOF) Laue technique. The microbial culture and crystal growth facilities which support the IMAGINE and MaNDi user programs are also described.