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The planarity of the peptide bond is important for the stability and structure formation of proteins. However, substantial distortion of peptide bonds has been reported in several high-resolution structures and computational analyses. To investigate the peptide bond planarity, including hydrogen atoms, we report a 1.2-Å resolution neutron structure of the oxidized form of high-potential iron-sulfur protein. This high-resolution neutron structure shows that the nucleus positions of the amide protons deviate from the peptide plane and shift toward the acceptors. The planarity of the H─N─C═O plane depends strongly on the pyramidalization of the nitrogen atom. Moreover, the orientation of the amide proton of Cys⁷⁵ is different in the reduced and oxidized states, possibly because of the electron storage capacity of the iron-sulfur cluster., タンパク質の立体構造解析に新たなモデルを提唱 --より正確な立体構造の観測や予測を実現! 生命科学研究の進展に寄与--. 京都大学プレスリリース. 2022-05-27.

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

Significance X-ray crystallography often fails to determine the positions of hydrogen atoms, which play crucial roles in enzymatic reactions. Despite many X-ray crystallographic studies, the reaction mechanism of copper-containing nitrite reductases (CuNIRs), which reduce nitrite using two protons, has been controversial. The high-resolution neutron structure of a CuNIR reveals the protonation states of catalytic residues and key water molecules, thus providing insights into the catalytic mechanism. The catalytic Cu is shown to be coordinated by a hydroxide ion and not water. Furthermore, the hydrogen-deuterium exchange ratio suggests that intramolecular electron transfer is involved in a hydrogen-bond jump. These observations are consistent with previous computational chemistry; therefore, our study forms a bridge between the structural biology and quantum chemistry of CuNIRs., Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Å resolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.

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 this article, the status of the STARGazer data-processing software and its data-processing algorithms are described in detail. The STARGazer data-processing software is used for neutron time-of-flight single-crystal diffraction data collected using the IBARAKI Biological Crystal Diffractometer., The STARGazer data-processing software is used for neutron time-of-flight (TOF) single-crystal diffraction data collected using the IBARAKI Biological Crystal Diffractometer (iBIX) at the Japan Proton Accelerator Research Complex (J-PARC). This software creates hkl intensity data from three-dimensional (x, y, TOF) diffraction data. STARGazer is composed of a data-processing component and a data-visualization component. The former is used to calculate the hkl intensity data. The latter displays the three-dimensional diffraction data with searched or predicted peak positions and is used to determine and confirm integration regions. STARGazer has been developed to make it easier to use and to obtain more accurate intensity data. For example, a profile-fitting method for peak integration was developed and the data statistics were improved. STARGazer and its manual, containing installation and data-processing components, have been prepared and provided to iBIX users. This article describes the status of the STARGazer data-processing software and its data-processing algorithms.

To improve the sensitivity of hydrogen detection using neutrons, high-pressure cooling of a relatively large protein single crystal and proton polarization of a protein polycrystalline state have been demonstrated as preliminary experiments., To improve the sensitivity of hydrogen detection using neutrons, a proton-polarization technique together with a high-pressure cooling method is necessary. The highest pressure (200 MPa) used in the experiment described here enabled relatively large protein crystals to be cooled without any cryoprotectants while retaining the protein structure, and it was confirmed that high-pressure-cooled crystals diffracted to nearly the same resolution as flash-cooled small crystals soaked with cryoprotectants. Dynamic nuclear polarization was used as a proton-polarization technique for protein crystals, and ∼300 mg polycrystalline protein doped with TEMPOL gave a maximum proton polarization of 22.3% at a temperature of 0.5 K in a 2.5 T magnetic field.

We plan to design a high-resolution biomacromolecule neutron time-of-flight diffractometer, which allows us to collect data from crystals with unit cells above 250 Å, in the materials and life science experimental facility at the Japan Proton Accelerator Research Complex. This new diffractometer can be used for a detailed analysis of large proteins such as membrane proteins and supermolecular complex. A quantitative comparison of the intensity and pulse width of a decoupled moderator (DM) against a coupled moderator (CM) considering the pulse width time resolution indicated that the DM satisfies the criteria for our diffractometer rather than the CM. The results suggested that a characteristic feature of the DM, i.e., narrow pulse width with a short tail, is crucial for the separation of Bragg reflections from crystals with large unit cells. On the other hand, it should be noted that the weak signals from the DM are buried under the high-level background caused by the incoherent scattering of hydrogen atoms, especially, in the case of large unit cells. We propose a profile-fitting integration method combined with the energy loss functions and a background subtraction method achieved by employing the statistics-sensitive nonlinear iterative peak-clipping algorithm.

The time-of-flight neutron single-crystal diffractometer iBIX at the next-generation neutron source J-PARC has been upgraded and is available for user experiments on protein samples in particular. Neutron structure analysis of a standard protein sample was carried out in order to evaluate the performance of iBIX., The IBARAKI biological crystal diffractometer, iBIX, is a high-performance time-of-flight neutron single-crystal diffractometer for elucidating mainly the hydrogen, protonation and hydration structures of biological macromolecules in various life processes. Since the end of 2008, iBIX has been available to users’ experiments supported by Ibaraki University. Since August 2012, an upgrade of the 14 existing detectors has begun and 16 new detectors have been installed for iBIX. The total measurement efficiency of the present diffractometer has been improved by one order of magnitude from the previous one with the increasing of accelerator power. In December 2012, commissioning of the new detectors was successful, and collection of the diffraction dataset of ribonucrease A as a standard protein was attempted in order to estimate the performance of the upgraded iBIX in comparison with previous results. The resolution of diffraction data, equivalence among intensities of symmetry-related reflections and reliability of the refined structure have been improved dramatically. iBIX is expected to be one of the highest-performance neutron single-crystal diffractometers for biological macromolecules in the world.

Fundamental trials to realise the proton polarization technique for detecting hydrogen with higher sensitivity in neutron protein crystallography are described., The isotope effect in conventional neutron protein crystallography (NPC) can be eliminated by the proton polarization technique (ppt). Furthermore, the ppt can improve detection sensitivity of hydrogen (relative neutron scattering length of hydrogen) by approximately eight times in comparison with conventional NPC. Several technical difficulties, however, should be overcome in order to perform the ppt. In this paper, two fundamental studies to realise ppt are presented: preliminary trials using high-pressure flash freezing has shown the advantage of making bulk water amorphous without destroying the single crystal; and X-ray diffraction and liquid-chromatography/mass-spectrometry analyses of standard proteins after introducing radical molecules into protein crystals have shown that radical molecules could be distributed non-specifically around proteins, which is essential for better proton polarization.

The neutron crystal structure of human transthyretin is presented., Transthyretin (TTR) is a tetrameric protein. TTR misfolding and aggregation are associated with human amyloid diseases. Dissociation of the TTR tetramer is believed to be the rate-limiting step in the amyloid fibril formation cascade. Low pH is known to promote dissociation into monomer and the formation of amyloid fibrils. In order to reveal the molecular mechanisms underlying pH sensitivity and structural stabilities of TTR, neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. Crystals for the neutron diffraction experiments were grown up to 2.5 mm3 for four months. The neutron crystal structure solved at 2.0 Å revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is involved in monomer–monomer and dimer–dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. Structural comparison with the X-ray crystal structure at acidic pH identified the three amino acid residues responsible for the pH sensitivity of TTR. Our neutron model provides insights into the molecular stability related to amyloidosis.

We developed and employed a profile fitting method for the peak integration of neutron time-of-flight diffraction data collected by the IBARAKI Biological Crystal Diffractometer (iBIX) at the Japan Proton Accelerator Research Complex (J-PARC) for protein ribonuclease A and α-thrombin single crystals. In order to determine proper fitting functions, four asymmetric functions were evaluated using strong intensity peaks. A Gaussian convolved with two back-to-back exponentials was selected as the most suitable fitting function, and a profile fitting algorithm for the integration method was developed. The intensity and structure refinement data statistics of the profile fitting method were compared to those of the summation integration method. It was clearly demonstrated that the profile fitting method provides more accurate integrated intensities and model structures than the summation integration method at higher resolution shells. The integration component with the profile fitting method has already been implemented in the iBIX data processing software STARGazer and its user manual has been prepared.

蛋白質結晶構造解析用の新型中性子回折装置である茨城県生命物質構造解析装置(iBIX)が大強度陽子加速器施設(J-PARC)の次世代中性子源に建設され，2008年12月から本格運転を開始した。2009年度には，14台の検出器，データ処理ソフトウェアの基本的な部分，20Kまでの低温吹付装置が利用可能となり，有機物標準結晶の測定及び構造解析に成功した。2009年12月からの120kW加速器運転より，産業界ユーザーの試料を含む複数の無機，有機化合物そして蛋白質結晶の構造解析用中性子測定がなされ，そのうちいくつかは水素や水和水の位置がわかる程度まで構造解析が進んでいる。

Ibaraki Prefectural Government together with Ibaraki University and Japan Atomic Energy Agency (JAEA) has almost finished constructing a time-of-flight (TOF) neutron diffractometer for biological macromolecules for industrial use at J-PARC, IBARAKI Biological Crystal Diffractometer (iBIX). Since 2009, Ibaraki University has been asked to operate this machine in order for users to do experiments by Ibaraki Prefecture. The diffractometer is designed to cover sample crystals which have their cell edges up to around 150 A. It is expected to measure more than 100 samples per year if they have 2 mm(3) in crystal volume, and to measure even around 0.1 mm(3) in crystal volume of biological samples. The efficiency of iBIX is also expected about 100 times larger than those of the present high performance diffractometers at JRR-3 in JAEA when 1MW power realizes in J-PARC. Since December 2008, iBIX has been open to users and several proteins and organic compounds were tested under 20 kW proton power of J-PARC. It was found that one of their proteins was diffracted up to 1.4 A in d-spacing, which was nearly comparable resolution to that of BIX-3 in JRR-3 when used the same crystal as at iBIX for reasonable exposure time. In May 2009, 14 detector units were set up. By the end of fiscal year 2009, the basic part of data reduction software will be finished and an equipment blowing low temperature gas to the sample will be installed with the cooperation of JAEA.

At the next generation neutron source in J-PARC, a new biological diffractometer, IBARAKI Biological Crystal Diffractometer (iBIX), is under construction, which is designed so that it can measure crystals with the cell edges measuring up to c.a. 150 A and the improved measurement efficiency will be more than 100 times larger than that of the present excellent diffractometer BIX-4 at JRR-3 in JAEA. In this paper the current status of the construction of iBIX is overviewed with expected high performances and new developments in chemistry and life science are predicted from recent results of neutron crystallography at JRR-3.

The structures of the melilite (three compounds) have been determined in the range from room temperature up to 773 K. The structures of incommensurate (IC) phase are characterized by bundles of four arrays of the six-coordinated Ca polyhedra with T 1 tetrahedra along the c-axis. The IC phases undergo the phase transition into the normal phases at elevated temperatures. The change of the modulated structure has been clarified by construction of the structures using the desired q-values and the modulation amplitudes determined at 293 K. The change of IC structures is characterized by variable distribution of bundle and octagonal arrangements of them. Large-scale regularities with sizes much larger than the modulation wavelength are also formed in the structures. The structure of commensurate phase of Ca2CoSi2O7 has been determined based on the results of simulation.

Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the Newton’s cradle - like proton relay pathway of the catalytic cycle. Amide-imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions., 形態: カラー図版あり, Physical characteristics: Original contains color illustrations, 資料番号: PA1610030000

A synchrotron radiation white X-ray diffraction of a Heusler-type Ni 2 . 1 8 Mn 0 . 8 2 Ga single crystal was observed by changing the temperature from 400 to 103 K. To investigate precisely the process of the martensitic transformation, changes in Laue spots during the transformation process were observed. The following phenomena were discovered: The single crystal at 300 Kb efore increasing temperature shows a tetragonal structure, the a- or b-axis of which is parallel to the direction of crystal growth. It shows a cubic Heusler structure at 400 K after heating. One of the cubic axes is parallel to the direction of crystal growth. With decreasing temperature, the crystal transforms into many small variants with tetragonal structures. One of the variants evolves so that the a- or b-axis is parallel to the direction of the crystal growth, and the orientation of the other variants can be represented by its rotation about the cubic axis. The rotation angle is at most a few degrees, which changes gradually with decreasing temperature. At 103 K the transformation is almost complete and many tetragonal variants are rearranged into one tetragonal structure, the a- or b-axis of which is parallel to the direction of crystal growth.

The neutron instruments suite, installed at the spallation neutron source of the Materials and Life Science Experimental Facility (MLF) at the Japan Proton Accelerator Research Complex (J-PARC), is reviewed. MLF has 23 neutron beam ports and 21 instruments are in operation for user programs or are under commissioning. A unique and challenging instrumental suite in MLF has been realized via combination of a high-performance neutron source, optimized for neutron scattering, and unique instruments using cutting-edge technologies. All instruments are/will serve in world-leading investigations in a broad range of fields, from fundamental physics to industrial applications. In this review, overviews, characteristic features, and typical applications of the individual instruments are mentioned.

Many studies on melilite group have been carried out in these two decades, and two significant features of the materials have been clarified so far. A number of descriptive and synthetic studies of melilite group have revealed that the compounds are highly tolerant of accommodating various combinations of elements in the structure. Another feature is the incommensurate structure-modulation found in melilites containing Ca as a major element. Structural studies with X-ray diffraction data and a treatment in the five-dimensional space clarified that formation and ordered arrangements of the six-coordinated Ca atoms play an important role to stabilize the long-range order in the modulated structures.

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The incommensurate modulation in a synthetic iron-bearing akermanite, Ca2(Mg, Fe )Si207, has been determined at room tempe~ature by the five-dimensional refinements of the structure. The basic structure is tetragonal P4 21m with unit-cell dimensions a =7.8679(3), c ~5.0144(2)A, V=31OAl(2)A3, 2=2, M=286.69, Dx =3.067Mg . m-3, MoKa with graphite monochromator, J1 =3.199mm-l, F(000)=284A9, Mg/(Mg+Fe)=0.554(6), R=0.065 for 770 unique reflections. The modulated structure is also tetragonal P~~~km, kt = 0.295(2)x (a*+b*), k2=0.295(2)x(-a*+b*), where kt. k2 are the wave vectors and a*, b* the reciprocal lattice vectors of the basic structure. R=0.136 for 3965 unique reflections. The modulation is caused mainly by the shifts of Ca and 0 atoms, and substitutional modulation at (Mg, Fe) sites was not detected in the present studies. Six-coordinated Ca forming a distorted oxygen octahedra exists in the modulated structure and most of them form clusters together with the flattened (Mg,Fe )04 tetrahedra. The number of the clusters in the present material is less than that in Co-akermanite. The reason may be attributed to the disorder of the modulation amplitudes and wavelength.

The crystallization-phase-diagram-guided method is effective for growing large protein crystals for neutron protein crystallography., Neutron protein crystallography (NPC) is a powerful tool for determining the hydrogen position and water orientation in proteins, but a much larger protein crystal is needed for NPC than for X-ray crystallography, and thus crystal preparation is a bottleneck. To obtain large protein crystals, it is necessary to know the properties of the target protein in the crystallization solution. Here, a crystal preparation method of fungal cellulase PcCel45A is reported, guided by the phase diagram. Nucleation and precipitation conditions were determined by sitting-drop vapor diffusion. Saturation and unsaturation conditions were evaluated by monitoring crystal dissolution, and a crystallization phase diagram was obtained. To obtain a large crystal, crystallization solution was prepared on a sitting bridge (diameter = 5 mm). Initial crystallization conditions were 40 µl of crystallization solution (40 mg ml−1 protein with 30.5% 3-methyl-1,5-pentanediol in 50 mM tris-HCl pH 8.0) with a 1000 µl reservoir (61% 3-methyl-1,5,-pentanediol in 50 mM tris-HCl pH 8.0) at 293 K. After the first crystal appeared, the concentration of precipitant in the reservoir solution was reduced to 60% to prevent formation of further crystals. Finally, we obtained a crystal of 6 mm3 volume (3 mm × 2 mm × 1 mm), which was suitable for neutron diffraction.

The isotope effect in conventional neutron protein crystallography (NPC) can be eliminated by the proton polarization technique (ppt) as an advanced NPC. Furthermore, the ppt can improve detection sensitivity of hydrogen (relative neutron scattering length of polarized proton) by approximately eight times in comparison with conventional NPC. Several technical difficulties, however, should be overcome in order to perform the ppt. In this poster, two developing fundamental studies to realize ppt will be presented; 1) radical doping into protein crystals that facilitates sample electron polarization, which was analyzed by X-ray crystallography, liquid-chromatography/mass-spectrometry (LC/MS) and electron spin resonance (ESR) measurement, 2) high-pressure flash freezing performed especially using a new machine of HPC-201 (ADC Inc.), which has the advantage of making bulk water amorphous without destroying the single large crystal, may easily realize the low temperature environment of crystal at around 1K. The former results were that radical molecules distributed non-specifically around proteins, and that they were included in protein crystal to some extent [1]. These are a favorable tendency for better proton polarization.