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

Etudes cristallographiques aux rayons X et aux neutrons du cytochrome P450cam et de la D-xylose isomerase

Les protons ont un rôle essentiel dans les mécanismes enzymatiques. Notre compréhension de ces processus peut donc grandement bénéficier de la localisation des hydrogènes aux sites actifs. Cette information peut-être obtenue par la cristallographie aux neutrons, aux résolutions modestes de 1.5 - 2.0 A. Cependant la cristallographie aux neutrons implique de nombreux challenges. De larges cristaux sont nécessaires pour palier aux faibles flux de neutrons disponibles. De plus, l'importante diffusion incohérente de l'hydrogène réduit considérablement le signal/bruit. Cela peut être contourné en deutérant l'échantillon, partiellement par trempage en solution deutérée ou complètement en produisant une protéine deutérée. La perdeutération offre un gain important en améliorant le signal/bruit d'un ordre de grandeur (permettant l'utilisation de cristaux plus petits) et en renforçant la visibilité des cartes neutroniques. Les cytochromes P450 sont impliqués dans de nombreux processus vitaux. Ils catalysent l'addition d'oxygène à des composés non-activés. La compréhension de leur mécanisme nécessite la connaissance de la position des hydrogènes au site actif. Pour bénéficier du gain offert par la perdeutération, le cytochrome P450cam perdeutéré a été produit. Son intégrité a été évaluée par FTIR et cristallographie aux rayons X. La D-xylose isomerase est une enzyme utilisée industriellement pour convertir la glucose en fructose. La structure neutronique de la D-xylose isomerase partiellement deutérée a été déterminée pour définir des détails critiques de son mécanisme enzymatique.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

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

Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

Metalloproteins perform a diverse array of redox-related reactions facilitated by the increased chemical functionality afforded by their metallocofactors. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that are responsible for the breakdown of recalcitrant polysaccharides via oxidative cleavage at the glycosidic bond. The activated copper-oxygen intermediates and their mechanism of formation remains to be established. Neutron protein crystallography which permits direct visualization of protonation states was used to investigate the initial steps of oxygen activation directly following active site copper reduction in Neurospora crassa LPMO9D. Herein, we cryo-trap an activated dioxygen intermediate in a mixture of superoxo and hydroperoxo states, and we identify the conserved second coordination shell residue His157 as the proton donor. Density functional theory (DFT) calculations indicate that both active site states are stable. The hydroperoxo formed is potentially an intermediate in the mechanism of hydrogen peroxide formation in the absence of substrate. We establish that the N-terminal amino group of the copper coordinating His1 remains doubly protonated directly following molecular oxygen reduction by copper. Aided by mining minima free energy calculations we establish His157 conformational flexibility in solution that is abolished by steric hindrance in the crystal. A neutron crystal structure of NcLPMO9D at low pH supports occlusion of the active site which prevents protonation of His157 at acidic conditions

An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase

The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed

The Structure of Sindbis Virus Produced from Vertebrate and Invertebrate Hosts as Determined by Small-Angle Neutron Scattering▿

The complex natural cycle of vectored viruses that transition between host species, such as between insects and mammals, makes understanding the full life cycle of the virus an incredibly complex problem. Sindbis virus, an arbovirus and prototypic alphavirus having an inner protein shell and an outer glycoprotein coat separated by a lipid membrane, is one example of a vectored virus that transitions between vertebrate and insect hosts. While evidence of host-specific differences in Sindbis virus has been observed, no work has been performed to characterize the impact of the host species on the structure of the virus. Here, we report the first study of the structural differences between Sindbis viruses grown in mammalian and insect cells, which were determined by small-angle neutron scattering (SANS), a nondestructive technique that did not decrease the infectivity of the Sindbis virus particles studied. The scattering data and modeling showed that, while the radial position of the lipid bilayer did not change significantly, it was possible to conclude that it did have significantly more cholesterol when the virus was grown in mammalian cells. Additionally, the outer protein coat was found to be more extended in the mammalian Sindbis virus. The SANS data also demonstrated that the RNA and nucleocapsid protein share a closer interaction in the mammalian-cell-grown virus than in the virus from insect cells