Macromolecular structure phasing by neutron anomalous diffraction.

In this report we show for the first time that neutron anomalous dispersion can be used in a practical manner to determine experimental phases of a protein crystal structure, providing a new tool for structural biologists. The approach is demonstrated through the use of a state-of-the-art monochromatic neutron diffractometer at the Institut Laue-Langevin (ILL) in combination with crystals of perdeuterated protein that minimise the level of hydrogen incoherent scattering and enhance the visibility of the anomalous signal. The protein used was rubredoxin in which cadmium replaced the iron at the iron-sulphur site. While this study was carried out using a steady-state neutron beam source, the results will be of major interest for capabilities at existing and emerging spallation neutron sources where time-of-flight instruments provide inherent energy discrimination. In particular this capability may be expected to offer unique opportunities to a rapidly developing structural biology community where there is increasing interest in the identification of protonation states, protein/water interactions and protein-ligand interactions - all of which are of central importance to a wide range of fundamental and applied areas in the biosciences.

Binding site asymmetry in human transthyretin: insights from a joint neutron and X-ray crystallographic analysis using perdeuterated protein.

Human transthyretin has an intrinsic tendency to form amyloid fibrils and is heavily implicated in senile systemic amyloidosis. Here, detailed neutron structural studies of perdeuterated transthyretin are described. The analyses, which fully exploit the enhanced visibility of isotopically replaced hydrogen atoms, yield new information on the stability of the protein and the possible mechanisms of amyloid formation. Residue Ser117 may play a pivotal role in that a single water molecule is closely associated with the γ-hydrogen atoms in one of the binding pockets, and could be important in determining which of the two sites is available to the substrate. The hydrogen-bond network at the monomer-monomer interface is more extensive than that at the dimer-dimer interface. Additionally, the edge strands of the primary dimer are seen to be favourable for continuation of the ß-sheet and the formation of an extended cross-ß structure through sequential dimer couplings. It is argued that the precursor to fibril formation is the dimeric form of the protein.

Glycyl-L-alanine: a multi-temperature neutron study.

Neutron diffraction data have been collected at 12, 50, 150 and 295 K for the dipeptide glycyl-L-alanine, C5H10N2O3, in order to obtain accurate positional and anisotropic displacement parameters for the H atoms. The values of these parameters serve as a benchmark for assessing the equivalent parameters obtained from a so-called Hirshfeld-atom refinement of X-ray diffraction data described elsewhere [Capelli et al. (2014). IUCrJ, 1, 361-379]. The flexibility of the glycyl-L-alanine molecule in the solid and the hydrogen-bonding interactions as a function of temperature are also considered.

CNN pincer ruthenium catalysts for hydrogenation and transfer hydrogenation of ketones: experimental and computational studies.

Reaction of [RuCl(CNN)(dppb)] (1-Cl) (HCNN=2-aminomethyl-6-(4-methylphenyl)pyridine; dppb=Ph2 P(CH2 )4 PPh2 ) with NaOCH2 CF3 leads to the amine-alkoxide [Ru(CNN)(OCH2 CF3 )(dppb)] (1-OCH2 CF3 ), whose neutron diffraction study reveals a short RuO⋅⋅⋅HN bond length. Treatment of 1-Cl with NaOEt and EtOH affords the alkoxide [Ru(CNN)(OEt)(dppb)]⋅(EtOH)n (1-OEt⋅n EtOH), which equilibrates with the hydride [RuH(CNN)(dppb)] (1-H) and acetaldehyde. Compound 1-OEt⋅n EtOH reacts reversibly with H2 leading to 1-H and EtOH through dihydrogen splitting. NMR spectroscopic studies on 1-OEt⋅n EtOH and 1-H reveal hydrogen bond interactions and exchange processes. The chloride 1-Cl catalyzes the hydrogenation (5 atm of H2 ) of ketones to alcohols (turnover frequency (TOF) up to 6.5×10(4) h(-1) , 40 °C). DFT calculations were performed on the reaction of [RuH(CNN')(dmpb)] (2-H) (HCNN'=2-aminomethyl-6-(phenyl)pyridine; dmpb=Me2 P(CH2 )4 PMe2 ) with acetone and with one molecule of 2-propanol, in alcohol, with the alkoxide complex being the most stable species. In the first step, the Ru-hydride transfers one hydrogen atom to the carbon of the ketone, whereas the second hydrogen transfer from NH2 is mediated by the alcohol and leads to the key "amide" intermediate. Regeneration of the hydride complex may occur by reaction with 2-propanol or with H2 ; both pathways have low barriers and are alcohol assisted.

L-Arabinose binding, isomerization, and epimerization by D-xylose isomerase: X-ray/neutron crystallographic and molecular simulation study.

D-xylose isomerase (XI) is capable of sugar isomerization and slow conversion of some monosaccharides into their C2-epimers. We present X-ray and neutron crystallographic studies to locate H and D atoms during the respective isomerization and epimerization of L-arabinose to L-ribulose and L-ribose, respectively. Neutron structures in complex with cyclic and linear L-arabinose have demonstrated that the mechanism of ring-opening is the same as for the reaction with D-xylose. Structural evidence and QM/MM calculations show that in the reactive Michaelis complex L-arabinose is distorted to the high-energy (5)S1 conformation; this may explain the apparent high KM for this sugar. MD-FEP simulations indicate that amino acid substitutions in a hydrophobic pocket near C5 of L-arabinose can enhance sugar binding. L-ribulose and L-ribose were found in furanose forms when bound to XI. We propose that these complexes containing Ni(2+) cofactors are Michaelis-like and the isomerization between these two sugars proceeds via a cis-ene-diol mechanism.

SPINE-compatible `carboloops': a new microshaped vitreous carbon sample mount for X-ray and neutron crystallography.

A novel vitreous carbon mount for macromolecular crystallography, suitable for neutron and X-ray crystallographic studies, has been developed. The technology described here is compatible both with X-ray and neutron cryo-crystallography. The mounts have low density and low background scattering for both neutrons and X-rays. They are prepared by laser cutting, allowing high standards of production quality, the ability to custom-design the mount to specific crystal sizes and large-scale production.

The self-assembling zwitterionic form of L-phenylalanine at neutral pH.

The title zwitterion (2S)-2-azaniumyl-1-hydroxy-3-phenylpropan-1-olate, C9H11NO2, also known as L-phenylalanine, was characterized using synchrotron X-rays. It crystallized in the monoclinic space group P21 with four molecules in the asymmetric unit. The 0.62 Šresolution structure is assumed to be closely related to the fibrillar form of phenylalanine, as observed by electron microscopy and electron diffraction. The structure exists in a zwitterionic form in which π-π stacking and hydrogen-bonding interactions are believed to form the basis of the self-assembling properties.

Nature of Si-H interactions in a series of ruthenium silazane complexes using multinuclear solid-state NMR and neutron diffraction.

Three new N-heterocyclic-silazane compounds, 1a-c, were prepared and employed as bidentate ligands to ruthenium, resulting in a series of [Ru(H){(κ-Si,N-(SiMe2-N-heterocycle)}3] complexes (3a-c) featuring the same RuSi3H motif. Detailed structural characterization of the RuSi3H complexes with X-ray diffraction, and in the case of triazabicyclo complex [Ru(H){κ-Si,N-(SiMe2)(C7H12N3)}3] (3a), neutron diffraction, enabled a reliable description of the molecular geometry. The hydride ligand of (3a) is located closer to two of the silicon atoms than it is to the third. Such a geometry differs from that of the previously reported complex [Ru(H){(κ-Si,N-(SiMe2)N(SiMe2H)(C5H4N)}3] (3d), also characterized by neutron diffraction, where the hydride was found to be equidistant from all three silicon atoms. A DFT study revealed that the symmetric and less regular isomers are essentially degenerate. Information on the dynamics and on the Ru···H···Si interactions was gained from multinuclear solid-state ((1)H wPMLG, (29)Si CP MAS, and 2D (1)H-(29)Si dipolar HETCOR experiments) and solution NMR studies. The corresponding intermediate complexes, [Ru{κ-Si,N-(SiMe2-N-heterocycle)}(η(4)-C8H12)(η(3)-C8H11)] (2a-c), involving a single silazane ligand were isolated and characterized by multinuclear NMR and X-ray diffraction. Protonation of the RuSi3H complexes was also studied. Reaction of 3a with NH4PF6 gave rise to [Ru(H)(η(2)-H -SiMe2)κ-N-(C7H12N3){κ-Si,N-(SiMe2)(C7H12N3)}2](+)[PF6](-)(4aPF6) which was isolated and characterized by NMR spectroscopy, X-ray crystallography, and DFT studies. The nature of the Si-H interactions in this silazane series was analyzed in detail.

A highly hydrated α-cyclodextrin/1-undecanol inclusion complex: crystal structure and hydrogen-bond network from high-resolution neutron diffraction at 20 K.

The monoclinic C2 crystal structure of an α-cyclodextrin/1-undecanol host-guest inclusion complex was solved using single-crystal neutron diffraction. Large high-quality crystals were specially produced by optimizing temperature-controlled growth conditions. The hydrate crystallizes in a channel-type structure formed by head-to-head dimer units of α-cyclodextrin molecules stacked like coins in a roll. The alkyl chain of the guest lipid is entirely embedded inside the tubular cavity delimited by the α-cyclodextrin dimer and adopts an all-trans planar zigzag conformation, while the alcohol polar head group is outside close to the α-cyclodextrin primary hydroxyl groups. The cyclodextrin dimer forms columns, which adopt a quasi-square arrangement much less compact than the quasi-hexagonal close packing already observed in the less hydrated α-cyclodextrin channel-type structures usually found with similar linear guests. The lack of compactness of this crystal form is related to the high number of interstitial water molecules. The replacement of 1-undecanol by 1-decanol does not modify the overall crystal structure of the hydrate as shown by additional X-ray diffraction investigations comparing the two host-guest assemblies. This is the first study that analyses the entire hydrogen-bonding network involved in the formation of a cyclodextrin dimer surrounded by its shell of water molecules.

Step-by-step introduction of silazane moieties at ruthenium: different extents of Ru-H-Si bond activation.

The coordination of pyridine-2-amino(methyl)dimethylsilane ligands to ruthenium has afforded access to a family of novel complexes that display multicenter Ru-H-Si interactions according to the number of incorporated ligands. The new complexes Ru[κ-Si,N-(SiMe2)N(Me)(C5H4N)](η(4)-C8H12)(η(3)-C8H11) (1), Ru2(µ-H)2(H)2[κ-Si,N-(SiMe2)N(Me)(C5H4N)]4 (2), and Ru(H)[κ-Si,N-(SiMe2)N(Me)(C5H4N)]3 (3) were isolated and fully characterized. The complexes exhibit different degrees of Si-H activation: complete Si-H cleavage, secondary interactions between the atoms (SISHA), and η(2)-Si-H coordination. Reversible protonation of 3 leading to the cationic complex [RuH{(η(2)-H-SiMe2)N(Me)κ-N-(C5H4N)}{κ-Si,N-(SiMe2)N(Me)(C5H4N)}2](+)[BAr(F)4](-) (5) was also demonstrated. The coordination modes in these systems were carefully studied with a combination of X-ray and neutron diffraction analysis, DFT geometry optimization, and multinuclear NMR spectroscopy.

Probing highly selective H/D exchange processes with a ruthenium complex through neutron diffraction and multinuclear NMR studies.

Deuterium labeling is a powerful way to gain mechanistic information in biology and chemistry. However, selectivity is hard to control experimentally, and labeled sites can be difficult to assign both in solution and in the solid state. Here we show that very selective high-deuterium contents can be achieved for the polyhydride ruthenium phosphine complex [RuH2(H2)2(PCyp3)2] (1) (PCyp3 = P(C5H9)3). The selectivity of the H/D exchange process is demonstrated by multinuclear NMR and neutron diffraction analyses. It has also been investigated through density functional theory (DFT) calculations. The reactions are performed under mild conditions at room temperature, and the extent of deuterium incorporation, involving selective C-H bond activation within the cyclopentyl rings of the phosphine ligands, can easily be tuned (solvent effects, D2 pressure). It is shown that D2 gas can inhibit the C-H/C-D exchange process.

The role of order-disorder transitions in the quest for molecular multiferroics: structural and magnetic neutron studies of a mixed valence iron(II)-iron(III) formate framework.

Neutron diffraction studies have been carried out to shed light on the unprecedented order-disorder phase transition (ca. 155 K) observed in the mixed-valence iron(II)-iron(III) formate framework compound [NH(2)(CH(3))(2)](n)[Fe(III)Fe(II)(HCOO)(6)](n). The crystal structure at 220 K was first determined from Laue diffraction data, then a second refinement at 175 K and the crystal structure determination in the low temperature phase at 45 K were done with data from the monochromatic high resolution single crystal diffractometer D19. The 45 K nuclear structure reveals that the phase transition is associated with the order-disorder of the dimethylammonium counterion that is weakly anchored in the cavities of the [Fe(III)Fe(II)(HCOO)(6)](n) framework. In the low-temperature phase, a change in space group from P31c to R3c occurs, involving a tripling of the c-axis due to the ordering of the dimethylammonium counterion. The occurrence of this nuclear phase transition is associated with an electric transition, from paraelectric to antiferroelectric. A combination of powder and single crystal neutron diffraction measurements below the magnetic order transition (ca. 37 K) has been used to determine unequivocally the magnetic structure of this Néel N-Type ferrimagnet, proving that the ferrimagnetic behavior is due to a noncompensation of the different Fe(II) and Fe(III) magnetic moments.

Inhibition of D-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography.

D-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni(2+) cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg(2+) ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni(2+) ions occupying the catalytic metal site (M2) were found at two locations, while Mg(2+) in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.

Uranium and thorium hydride complexes as multielectron reductants: a combined neutron diffraction and quantum chemical study.

The unusual uranium reaction system in which uranium(4+) and uranium(3+) hydrides interconvert by formal bimetallic reductive elimination and oxidative addition reactions, [(C(5)Me(5))(2)UH(2)](2) (1) ⇌ [(C(5)Me(5))(2)UH](2) (2) + H(2), was studied by employing multiconfigurational quantum chemical and density functional theory methods. 1 can act as a formal four-electron reductant, releasing H(2) gas as the byproduct of four H(2)/H(-) redox couples. The calculated structures for both reactants and products are in good agreement with the X-ray diffraction data on 2 and 1 and the neutron diffraction data on 1 obtained under H(2) pressure as part of this study. The interconversion of the uranium(4+) and uranium(3+) hydride species was calculated to be near thermoneutral (~-2 kcal/mol). Comparison with the unknown thorium analogue, [(C(5)Me(5))(2)ThH](2), shows that the thorium(4+) to thorium(3+) hydride interconversion reaction is endothermic by 26 kcal/mol.

(+)-syn-Benzotriborneol an enantiopure C3-symmetric receptor for water.

(+)-syn-Benzotriborneol forms stable complexes with one molecule of water. This is due to the ability of the host to form three hydrogen bonds with water, to act simultaneously as a hydrogen-bond acceptor and donor, and to a perfect geometrical match between the pair. We report experimental (X-ray and neutron diffraction, VT NMR, DSC, TGA) and stereochemical studies carried out to elucidate and quantify the molecular and thermodynamic aspects of this supramolecular complex.

Preliminary neutron crystallographic study of human transthyretin.

Preliminary studies of perdeuterated crystals of human transthyretin (TTR) have been carried out using the LADI-III and D19 diffractometers at the Institut Laue-Langevin in Grenoble. The results demonstrate the feasibility of a full crystallographic analysis to a resolution of 2.0 Å using Laue diffraction and also illustrate the potential of using monochromatic instruments such as D19 for higher resolution studies where larger crystals having smaller unit cells are available. This study will yield important information on hydrogen bonding, amino-acid protonation states and hydration in the protein. Such information will be of general interest for an understanding of the factors that stabilize/destabilize TTR and for the design of ligands that may be used to counter TTR amyloid fibrillogenesis.

Molecular heterometallic hydride clusters composed of rare-earth and d-transition metals.

Heteromultimetallic hydride clusters containing both rare-earth and d-transition metals are of interest in terms of both their structure and reactivity. However, such heterometallic complexes have not yet been investigated to a great extent because of difficulties in their synthesis and structural characterization. Here, we report the synthesis, X-ray and neutron diffraction studies, and hydrogen addition and release properties of a family of rare-earth/d-transition-metal heteromultimetallic polyhydride complexes of the core structure type 'Ln(4)MH(n)' (Ln = Y, Dy, Ho; M = Mo, W; n = 9, 11, 13). Monitoring of hydrogen addition to a hydride cluster such as [{(C(5)Me(4)SiMe(3))Y}(4)(µ-H)(9)Mo(C(5)Me(5))] in a single-crystal to single-crystal process by X-ray diffraction has been achieved for the first time. Density functional theory studies reveal that the hydrogen addition process is cooperatively assisted by the Y/Mo heteromultimetallic sites, thus offering unprecedented insight into the hydrogen addition and release process of a metal hydride cluster.

The structure of water in p-sulfonatocalix[4]arene.

Tetrasodium p-sulfonatocalix[4]arene exists as a hydrate with approximately 14 water molecules and has three polymorphic modifications, all of which contain a water molecule in the molecular cavity that is engaged in OH···π interactions. Single-crystal neutron structures are reported for two of these three forms and reveal a "compressed" water molecule with short OH bonds. Partial atomic charges and hardness analysis (PACHA) calculations based on the neutron coordinates give an OH···π interaction energy of 6.9-7.5 kJ mol(-1). The PACHA analysis also reveals the dominance of the charge-assisted hydrogen bonds from the Na(+)-coordinated water molecules. The instability of the crystal towards dehydration can be traced to an uncoordinated lattice water site. The remarkable calixarene-Na(+)-hydrate motif is conserved almost unchanged across all three polymorphs. A single-crystal neutron structure is also reported for pentasodium p-sulfonatocalix[4]arene·12H(2)O, which exhibits an intracavity water molecule that is engaged in both OH···π and OH···O hydrogen bonding. The shorter covalent bond to the hydrogen atom that forms the interaction with the aromatic ring is again apparent.

Square-planar iridium(II) and iridium(III) amido complexes stabilized by a PNP pincer ligand.

Squaring the circle: the novel dienamido pincer ligand N(CHCHPtBu(2))(2)(-) affords the isolation of the unusual square-planar iridium(II) and iridium(III) amido complexes [IrCl{N(CHCHPtBu(2))(2)}](n) (n=0 (1), +1 (2)). In contrast, the corresponding iridium(I) complex of the redox series (n=-1) is surprisingly unstable. The diamagnetism of 2 is attributed to strong N→Ir π donation.

High-resolution neutron crystallographic studies of the hydration of the coenzyme cob(II)alamin.

The hydration of the coenzyme cob(II)alamin has been studied using high-resolution monochromatic neutron crystallographic data collected at room temperature to a resolution of 0.92 Å on the original D19 diffractometer with a prototype 4° × 64° detector at the high-flux reactor neutron source run by the Institute Laue-Langevin. The resulting structure provides hydrogen-bonding parameters for the hydration of biomacromolecules to unprecedented accuracy. These experimental parameters will be used to define more accurate force fields for biomacromolecular structure refinement. The presence of a hydrophobic bowl motif surrounded by flexible side chains with terminal functional groups may be significant for the efficient scavenging of ligands. The feasibility of extending the resolution of this structure to ultrahigh resolution was investigated by collecting time-of-flight neutron crystallographic data during commissioning of the TOPAZ diffractometer with a prototype array of 14 modular 2° × 21° detectors at the Spallation Neutron Source run by Oak Ridge National Laboratory.