National Synchrotron Light Source II

High-performance electrocatalytic CO ₂ reduction to CO using Ni single-atom catalyst in an anion membrane electrode assembly.

Abstract Regioregular poly(3‐hexyl thiophene) (RR P3HT) is drop‐cast to fabricate field‐effect transistor (FET) devices from different solvents with different boiling points and solubilities for RR P3HT, such as methylene chloride, toluene, tetrahydrofuran, and chloroform. A Petri dish is used to cover the solution, and it takes less than 30 min for the solvents to evaporate at room temperature. The mesoscale crystalline morphology of RR P3HT thin films can be manipulated from well‐dispersed nanofibrils to well‐developed spherulites by changing solution processing conditions. The morphological correlation with the charge‐carrier mobility in RR P3HT thin‐film transistor (TFT) devices is investigated. The TFT devices show charge‐carrier mobilities in the range of 10 –4 ∼ 10 –2 cm 2 V –1 s –1 depending on the solvent used, although grazing‐incidence X‐ray diffraction (GIXD) reveals that all films develop the same π–π‐stacking orientation, where the <100>‐axis is normal to the polymer films. By combining results from atomic force microscopy (AFM) and GIXD, it is found that the morphological connectivity of crystalline nanofibrils and the <100>‐axis orientation distribution of the π–π‐stacking plane with respect to the film normal play important roles on the charge‐carrier mobility of RR P3HT for TFT applications.

The photoemission line shapes of the optimally doped cuprate Bi(2)Sr(2)CaCu(2)O(8+delta) were studied in the direction of a node in the superconducting order parameter by means of very high resolution photoemission spectroscopy. The peak width or inverse lifetime of the excitation displays a linear temperature dependence, independent of binding energy, for small energies, and a linear energy dependence, independent of temperature, for large binding energies. This behavior is unaffected by the superconducting transition, which is an indication that the nodal states play no role in the superconductivity. Temperature-dependent scaling suggests that the system displays quantum critical behavior.

Metal partitioning in ferrihydrite suspensions may reach equilibrium only after a long reaction time. To determine key factors controlling the kinetics, we measured Cu and Pb uptake as a function of ferrihydrite morphology, reaction temperature, metal competition, and fulvic acid concentration over a period of 2 months. X-ray microscopy, which was used to probe ferrihydrite morphology in suspension, showed that drying irreversibly converted the gellike structure of fresh precipitate into dense aggregates. These dense aggregates sorbed Cu and Pb much slower than the gel. Temperature had a more pronounced effect on the kinetics of metal uptake by ferrihydrite gel than by dense ferrihydrite. Independently of treatment and time, Cu and Pb were bound to the ferrihydrite surface byformation of edge-sharing inner-sphere sorption complexes as confirmed by X-ray absorption fine-structure (XAFS) spectroscopy. This invariable binding mechanism, together with the observed effects of morphology and temperature, are in line with surface diffusion limiting the slow sorption process. The quantification of diffusion-limited surface sites in soils and sediments and the subsequent estimation of the effect of reaction time and temperature will be a challenge for properly predicting the fate of metals in the environment.

Synchrotron x-ray reflectivity is used to study the interface between bulk water and bulk n-alkanes with carbon numbers 6 through 10, 12, 16, and 22. For all interfaces, except the water-hexane interface, the interfacial width disagrees with the prediction from capillary-wave theory. The variation of interfacial width with carbon number can be described by combining the capillary-wave prediction for the width with a contribution from intrinsic structure. This intrinsic structure is determined by the gyration radius for the shorter alkanes and by the bulk correlation length for the longer alkanes.

The methane activation and methane dry reforming reactions were studied and compared over 4 wt % Ni/CeO2 and 4 wt % Ni/CeZrO2 (containing 20 wt % Zr) catalysts. Upon the incorporation of Zr into the ceria support, the catalyst exhibited a significantly improved activity and H2 selectivity. To understand the effects of the Zr dopant on Ni and CeO2 during the dry reforming of methane (DRM) reaction and to probe the structure–reactivity relationship underlying the enhanced catalytic performance of the mixed-oxide system, in situ time-resolved X-ray diffraction (TR-XRD), X-ray absorption fine structure (XAFS), and ambient-pressure X-ray photoelectron spectroscopy (AP-XPS) were employed to characterize the catalysts under reaction conditions. TR-XRD and AP-XPS indicate that ceria–zirconia supported Ni (Ni/CeZrO2) is of higher reducibility than the pure ceria supported Ni (Ni/CeO2) upon the reaction with pure CH4 or for the methane dry reforming reaction. The active state of Ni/CeZrO2 under optimum DRM conditions (700 °C) was identified as Ni0, Ce3+/Ce4+, and Zr4+. The particle size of both nickel and the ceria support under reaction conditions was analyzed by Rietveld refinement and extended XAFS fitting. Zr in the ceria support prevents particle sintering and maintains small particle sizes for both metallic nickel and the partially reduced ceria support under reaction conditions through a stronger metal–support interaction. Additionally, Zr prevents Ni migration from the surface into ceria forming a Ce1–xNixO2–y solid solution, which is seen in Ni/CeO2, thus helping to preserve the active Ni0 on the Ni/CeZrO2 surface.

An integral LiMn _1.5 Ni _0.5 O ₄ shell completely wets ∼10 μm LiCoO ₂ single crystals to cut off global oxygen migration and enables >4.6 V cycling.

Root uptake, translocation, and distribution of engineered nanoparticles by plants are dependent on both plant species and nanoparticle surface charge.

We have determined the electronic and atomic structure of N doped TiO2 using a combination of hard x-ray photoelectron spectroscopy and first-principles density functional theory calculations. Our results reveal that N doping of TiO2 leads to the formation of oxygen vacancies and the combination of both N impurity and oxygen vacancies accounts for the observed visible light catalytic behavior of N doped TiO2.

-MIL-53(Al) in a diamond anvil cell under different pressurization media. Systematic refinements of the obtained powder patterns demonstrate that these materials expand along a specific direction while undergoing total volume reduction under an increase in hydrostatic pressure. The results confirm for the first time the Negative Linear Compressibility behaviour of this family of materials recently predicted from quantum chemical calculations.

The focusing performance of a multilayer Laue lens (MLL) with 43.4 μm aperture, 4 nm finest zone width and 4.2 mm focal length at 12 keV was characterized with X-rays using ptychography method. The reconstructed probe shows a full-width-at-half-maximum (FWHM) peak size of 11.2 nm. The obtained X-ray wavefront shows excellent agreement with the dynamical calculations, exhibiting aberrations less than 0.3 wave period, which ensures the MLL capable of producing a diffraction-limited focus while offering a sufficient working distance. This achievement opens up opportunities of incorporating a variety of in-situ experiments into ultra high-resolution X-ray microscopy studies.

We have determined the Ti and O components of the rutile TiO2 valence band using the method of site-specific x-ray photoelectron spectroscopy. Comparisons with calculations based on pseudopotentials within the local density approximation reveal the hybridization of the Ti 3d, 4s, and 4p states, and the O 2s and 2p states on each site. These chemical effects are observed due to the large differences between the angular-momentum dependent matrix elements of the photoelectron process.

For the first time, sulfonylated polymers of intrinsic microporosity (PIMs) are exploited for high- <italic>κ</italic> , high-temperature, and low-loss gate dielectric applications.

Surface reconstruction reactions that would determine the Li/Ni ordering in Ni-rich layered materials are investigated.

<italic>Operando</italic> , spatiotemporal resolved synchrotron X-ray fluorescence mapping measurements on a custom aqueous Zn/α-MnO ₂ cell provided direct, quantitative evidence of a Mn dissolution-deposition faradaic mechanism that governs the electrochemistry.

The synchrotron x-ray near total external fluorescence technique is used to measure the concentration profiles of elements near a sample surface along its surface normal. This novel nondestructive technique is capable of providing information about the concentration profiles on a length scale of tens to hundreds of angstroms from the sample surface. It does not require vacuum and can be applied at ambient pressures. Concentration profiles of virtually all the elements in the periodic table can be investigated using this technique. The lower limit of detection is approximately equivalent to (1)/(10) of a monolayer coverage. The technique is particularly useful in applications where the fluorescence signal originating from a thin surface layer can be normalized against that originating from the same element in the bulk of the sample. The experimental procedure and the data analysis are demonstrated using experimental results of metal-ion segregation from a liquid solution to a liquid/air interface.

The assembly of individual molecules into hierarchical structures is a promising strategy for developing three-dimensional materials with properties arising from interaction between the individual building blocks. Virus capsids are elegant examples of biomolecular nanostructures, which are themselves hierarchically assembled from a limited number of protein subunits. Here, we demonstrate the bio-inspired modular construction of materials with two levels of hierarchy: the formation of catalytically active individual virus-like particles (VLPs) through directed self-assembly of capsid subunits with enzyme encapsulation, and the assembly of these VLP building blocks into three-dimensional arrays. The structure of the assembled arrays was successfully altered from an amorphous aggregate to an ordered structure, with a face-centered cubic lattice, by modifying the exterior surface of the VLP without changing its overall morphology, to modulate interparticle interactions. The assembly behavior and resultant lattice structure was a consequence of interparticle interaction between exterior surfaces of individual particles and thus independent of the enzyme cargos encapsulated within the VLPs. These superlattice materials, composed of two populations of enzyme-packaged VLP modules, retained the coupled catalytic activity in a two-step reaction for isobutanol synthesis. This study demonstrates a significant step toward the bottom-up fabrication of functional superlattice materials using a self-assembly process across multiple length scales and exhibits properties and function that arise from the interaction between individual building blocks.

Human UHRF1 (ubiquitin-like PHD and RING finger 1) functions to maintain CpG DNA methylation patterns through DNA replication by co-localizing with the DNA methyltransferase DNMT1 at chromatin in mammals. Recent studies show that UHRF1 binds selectively to hemimethylated CpG via its conserved SRA (SET- and RING finger-associated) domain. However, the underlying molecular mechanism is not known. Here, we report a 1.95Å resolution crystal structure of the SRA domain of human UHRF1. Using NMR structure-guided mutagenesis, electrophoretic mobility shift assay, and fluorescence anisotropy analysis, we determined key amino acid residues for methyl-DNA binding that are conserved in the SRA domain. Human UHRF1 (ubiquitin-like PHD and RING finger 1) functions to maintain CpG DNA methylation patterns through DNA replication by co-localizing with the DNA methyltransferase DNMT1 at chromatin in mammals. Recent studies show that UHRF1 binds selectively to hemimethylated CpG via its conserved SRA (SET- and RING finger-associated) domain. However, the underlying molecular mechanism is not known. Here, we report a 1.95Å resolution crystal structure of the SRA domain of human UHRF1. Using NMR structure-guided mutagenesis, electrophoretic mobility shift assay, and fluorescence anisotropy analysis, we determined key amino acid residues for methyl-DNA binding that are conserved in the SRA domain. Almost all DNA methylation in the mammalian genome occurs in the CpG dinucleotide motif carried out by the DNA methyltransferase DNMT1 and is maintained through DNA replication (1.Bernstein B.E. Meissner A. Lander E.S. Cell. 2007; 128: 669-681Abstract Full Text Full Text PDF PubMed Scopus (1731) Google Scholar). As a heritable epigenetic mark, CpG methylation works in concert with histone modifications to control gene transcriptional silencing and heterochromatin formation (2.Johnson L. Bostick M. Zhang X. Kraft E. Henderson I. Callis J. Jacobsen S. Curr. Biol. 2007; 17: 379-384Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 3.Jaenisch R. Bird A. Nat. Genet. 2003; 33: 245-254Crossref PubMed Scopus (4838) Google Scholar). Studies show that the multidomain human protein UHRF1 (also known as ICBP90 in human and Np95 in mouse) functions to maintain CpG methylation patterns by co-localizing with DNMT1 (4.Bronner C. Achour M. Arima Y. Chataigneau T. Saya H. Schini-Kerth V.B. Pharmacol. Ther. 2007; 115: 419-434Crossref PubMed Scopus (143) Google Scholar, 5.Achour M. Jacq X. Ronde P. Alhosin M. Charlot C. Chataigneau T. Jeanblanc M. Macaluso M. Giordano A. Hughes A.D. Schini-Kerth V.B. Bronner C. Oncogene. 2008; 27: 2187-2197Crossref PubMed Scopus (139) Google Scholar) and the histone-lysine deacetylase HDAC1 (6.Unoki M. Nishidate T. Nakamura Y. Oncogene. 2004; 23: 7601-7610Crossref PubMed Scopus (243) Google Scholar). More recent studies show that UHRF1 has E3 ubiquitin ligase activity (7.Citterio E. Papait R. Nicassio F. Vecchi M. Gomiero P. Mantovani R. Di Fiore P. Bonapace I. Mol. Cell. Biol. 2004; 24: 2526-2535Crossref PubMed Scopus (163) Google Scholar) and can also selectively recognize methylated histone H3 at lysine 9 by its PHD finger (4.Bronner C. Achour M. Arima Y. Chataigneau T. Saya H. Schini-Kerth V.B. Pharmacol. Ther. 2007; 115: 419-434Crossref PubMed Scopus (143) Google Scholar, 8.Karagianni P. Amazit L. Qin J. Wong J. Mol. Cell. Biol. 2008; 28: 705-717Crossref PubMed Scopus (201) Google Scholar) and hemimethylated CpG by its SRA domain (6.Unoki M. Nishidate T. Nakamura Y. Oncogene. 2004; 23: 7601-7610Crossref PubMed Scopus (243) Google Scholar, 9.Bostick M. Kim J. Estève P. Clark A. Pradhan S. Jacobsen S. Science. 2007; 317: 1760-1764Crossref PubMed Scopus (1033) Google Scholar, 10.Sharif J. Muto M. Takebayashi S. Suetake I. Iwamatsu A. Endo T.A. Shinga J. Mizutani-Koseki Y. Toyoda T. Okamura K. Tajima S. Mitsuya K. Okano M. Koseki H. Nature. 2007; 450: 908-912Crossref PubMed Scopus (973) Google Scholar), thus bridging two important epigenetic marks in DNA and histones for functional regulation of gene silencing and pericentromeric heterochromatin formation (11.Papait R. Pistore C. Grazini U. Babbio F. Cogliati S. Pecoraro D. Brino L. Morand A.L. Dechampesme A.M. Spada F. Leonhardt H. McBlane F. Oudet P. Bonapace I.M. Mol. Biol. Cell. 2008; 19: 3554-3563Crossref PubMed Scopus (61) Google Scholar, 12.Woo H. Pontes O. Pikaard C. Richards E. Genes Dev. 2007; 21: 267-277Crossref PubMed Scopus (155) Google Scholar, 13.Liu S. Yu Y. Ruan Y. Meyer D. Wolff M. Xu L. Wang N. Steinmetz A. Shen W.H. Plant J. 2007; 52: 914-926Crossref PubMed Scopus (37) Google Scholar). Given its multiple functionality in epigenetic gene regulation, it is not surprising that human UHRF1 is found to be overexpressed in many different forms of human cancers, including breast (6.Unoki M. Nishidate T. Nakamura Y. Oncogene. 2004; 23: 7601-7610Crossref PubMed Scopus (243) Google Scholar, 14.Hopfner R. Mousli M. Oudet P. Bronner C. Anticancer Res. 2002; 22: 3165-3170PubMed Google Scholar, 15.Mousli M. Hopfner R. Abbady A.Q. Monte D. Jeanblanc M. Oudet P. Louis B. Bronner C. Br. J. Cancer. 2003; 89: 120-127Crossref PubMed Scopus (123) Google Scholar, 16.Jenkins Y. Markovtsov V. Lang W. Sharma P. Pearsall D. Warner J. Franci C. Huang B. Huang J. Yam G.C. Vistan J.P. Pali E. Vialard J. Janicot M. Lorens J.B. Payan D.G. Hitoshi Y. Mol. Biol. Cell. 2005; 16: 5621-5629Crossref PubMed Scopus (150) Google Scholar), cervical (17.Lorenzato M. Caudroy S. Bronner C. Evrard G. Simon M. Durlach A. Birembaut P. Clavel C. Hum. Pathol. 2005; 36: 1101-1107Crossref PubMed Scopus (71) Google Scholar), and prostate (16.Jenkins Y. Markovtsov V. Lang W. Sharma P. Pearsall D. Warner J. Franci C. Huang B. Huang J. Yam G.C. Vistan J.P. Pali E. Vialard J. Janicot M. Lorens J.B. Payan D.G. Hitoshi Y. Mol. Biol. Cell. 2005; 16: 5621-5629Crossref PubMed Scopus (150) Google Scholar) cancers; pancreatic adenocarcinomas (18.Crnogorac-Jurcevic T. Gangeswaran R. Bhakta V. Capurso G. Lattimore S. Akada M. Sunamura M. Prime W. Campbell F. Brentnall T.A. Costello E. Neoptolemos J. Lemoine N.R. Gastroenterology. 2005; 129: 1454-1463Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar); rhabdomyosarcomas (19.Schaaf G.J. Ruijter J.M. van Ruissen F. Zwijnenburg D.A. Waaijer R. Valentijn L.J. Benit-Deekman J. van Kampen A.H. Baas F. Kool M. FASEB J. 2005; 19: 404-406PubMed Google Scholar); and gliomas (20.Oba-Shinjo S.M. Bengtson M.H. Winnischofer S.M. Colin C. Vedoy C.G. de Mendonca Z. Marie S.K. Sogayar M.C. Mol. Brain Res. 2005; 140: 25-33Crossref PubMed Scopus (38) Google Scholar). However, molecular mechanistic understanding of UHR1 is limited. In an effort to determine its structural basis of hemimethylated DNA recognition, we solved a high resolution crystal structure of the SRA domain from human UHRF1. By using NMR structure-guided analysis and mutagenesis, we further identified the amino acid residues that are important for SRA domain recognition of hemimethylated DNA. Protein Preparation—The SRA domain (residues 414–617) of human UHRF1 was cloned into the pET28a plasmid as an N-terminal His6-tagged fusion protein. The fusion protein was expressed in Escherichia coli (strain BL21(DE3)). Uniformly 15N- and 15N/13C-labeled proteins were prepared by growing bacteria in minimal medium with 15NH4Cl and/or [13C6]glucose as the sole nitrogen and carbon sources. Deuterated protein was generated by cell growth in 90% 2H2O. The SRA domain proteins were purified by nickel-nitrilotriacetic acid affinity and size exclusion columns followed by Mono S ion exchange chromatography after thrombin cleavage of the His6 tag. Protein NMR samples (∼0.5 mm) were prepared in 50 mm sodium phosphate buffer (pH 6.5) containing 150 mm NaCl and 2 mm dithiothreitol-d10 in H2O/2H2O (9:1) or 2H2O. DNA Preparation—High pressure liquid chromatography-purified 5′-fluorescein-labeled, 5′-biotinylated, unmodified or hemimethylated CpG oligonucleotides were purchased from Fisher (supplemental Table 1). The lyophilized oligonucleotides were resuspended to 2.0 mm in 10 mm Tris-HCl (pH 8.0) containing 100 mm NaCl. Complementary strands were mixed in equal molar amounts and annealed by heating to 368 K and then cooled to room temperature. Site-directed Mutagenesis—Human UHRF1 SRA domain mutants were generated using the QuikChange kit (Stratagene) and cloned into the pGEX6p1 vector as an N-terminal glutathione S-transferase fusion protein. The presence of appropriate mutations was confirmed by DNA sequencing. NMR Spectroscopy—All NMR spectra were acquired at 293 K on an 800, 600, or 500 MHz NMR spectrometer. 1H, 13C, and 15N backbone resonances of the protein were assigned with three-dimensional deuterium-decoupled triple-resonance HNCA, HN(CA)CB, HN(COCA)CB, and HN(CO)-CACB spectra recorded on a uniformly 15N/13C-labeled and fractionally deuterated protein (21.Yamazaki T. Lee W. Arrowsmith C.H. Mahandiram D.R. Kay L.E. J. Am. Chem. Soc. 1994; 116: 11655-11666Crossref Scopus (503) Google Scholar). DNA titration was performed by recording a series of two-dimensional 15N heteronuclear single quantum coherence spectra on uniformly 15N-labeled SRA domain (∼0.5 mm) in the presence of different amounts of DNA oligonucleotides ranging from 0 to 0.05 mm. Crystallization—Native crystals of the UHRF1 SRA domain were obtained at 293 K with the vapor diffusion hanging drop method by mixing 1 μl of the protein solution with 1 μl of crystallization solution (100 mm Tris-HCl (pH 8.0) containing 200 mm NaCl, 1 m ammonium sulfate, and 15% glycerol). For initial phasing, sodium selenate (Na2SeO4) heavy atom derivative was prepared by soaking native SRA domain protein crystals in mother liquor solution in which the ammonium sulfate was substituted with sodium selenate while maintaining all remaining components constant. The crystal soaking was carried out for at least 1 h at 293 K to ensure SO4/SeO4 substitution. All diffraction data were measured at 100 K at Brookhaven National Laboratory beamlines X4C and X6A. The heavy atom soaked crystal and the native crystal diffracted to 3.0 and 1.95 Å, respectively. All data were processed with HKL-2000 (22.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38777) Google Scholar). The crystals belong to space group P3121 with unit cell dimensions of a = b = 65.6 Å, c = 95.4 Å, and α = β = γ = 120°. During the phasing process, one family protein crystal structure (Protein Data Bank code 3BI7) was released, which was used as a molecular replacement template. The molecular replacement solution was used subsequently for the structure refinement to 1.95 Å using REFMAC5 (23.Pannu N.S. Murshudov G.N. Dodson E.J. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 1285-1294Crossref PubMed Scopus (0) Google Scholar) and COOT (24.Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (24326) Google Scholar). X-ray data collection and refinement statistics are listed in Table 1.TABLE 1Crystallographic data collection and refinement statisticsData collection Wavelength (Å)/energy (keV)0.9192/12.67 Space groupP3121 Resolution limits (Å)20.00-1.95 (2.02-1.95) Completeness (%)aNumbers in parentheses refer to the highest resolution shell.98.7 (98.9) Average I/σI13.7 (2.5) Rmerge (%)bRmerge = Σ|I — 〈I〉|/ΣI, where I is the integrated intensity of a given intensity.8.1 (35.3) Average mosaicity0.35° Total reflections53,795 Unique reflections17,612 (1,739)Refinement Resolution (Å)20.0-1.95 (2.00-1.95) Rcryst/Rfree (%)cRcryst = Σ||Fo| — |Fc||/Σ|Fo|., dRfree was calculated using 10% random data omitted from the refinement.19.9/25.3 (21.9/27.0) r.m.s.d.er.m.s.d., root mean square deviation; GOL, glycerol; DPI, diffraction precision indicator. bond length (Å)/angle0.017/1.608° Non-H atoms, protein/water/ligands1,662/135/2 SO4 + 1 GOL B (overall, protein, water, ligands) (Å2)19.9/19.4/24.8/26.3 Coordinate error DPI (Å)0.152 Ramachandran plot quality, favored/additional/disallowed (%)89.5/10.5/0.0a Numbers in parentheses refer to the highest resolution shell.b Rmerge = Σ|I — 〈I〉|/ΣI, where I is the integrated intensity of a given intensity.c Rcryst = Σ||Fo| — |Fc||/Σ|Fo|.d Rfree was calculated using 10% random data omitted from the refinement.e r.m.s.d., root mean square deviation; GOL, glycerol; DPI, diffraction precision indicator. Open table in a new tab Fluorescence Anisotropy—The SRA domain binding affinity for 5′-fluorescein-labeled oligonucleotides was determined at 293 K using a TECAN Safire fluorescence reader with excitation at 470 nm and emission at 525 nm. The 5′-fluorescein-labeled DNA (5 nm) was added to a series of the wild-type or mutant SRA domain proteins with varying concentrations of 5 nm to 0.5 mm. Anisotropy values were referenced against a blank sample of buffer (20 mm Tris-HCl (pH 7.5) containing 50 mm NaCl and 1 mm dithiothreitol) at the beginning of each experiment to account for background correction. The anisotropy data were fitted by nonlinear least-squares regression to the following equation: A = Amin + ((E + D + Kd) – ((E + D + Kd)2 – 4DE)1/2)(Amax – Amin)/(2D), where A is the anisotropy, E is the total protein concentration, D is the total DNA concentration, Amin is the anisotropy of free DNA, Amax is the anisotropy of the DNA-protein complex, and Kd is the dissociation constant. A 1:1 stoichiometry for the DNA-protein complex was assumed. Electrophoretic Mobility Shift Assay—The SRA domain/5′-biotin-labeled DNA binding was carried out in 10 mm Tris buffer (pH 7.5) containing 50 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 0.05% Nonidet P-40, 2.5% glycerol, 50 ng/μl poly(dI-dC), and varying amounts of protein (0–15 μg) and/or biotin-labeled methyl-DNA (2–10 ng). The reaction mixtures were incubated at 296 K for 40 min and then electrophoresed on a 10% Tris borate/EDTA gel (Invitrogen) at 100 V for 1.5 h in 100 mm Tris borate/EDTA buffer. The reactions were transferred to a nylon membrane. The biotin-labeled DNA was detected with the LightShift chemiluminescent electrophoretic mobility shift assay kit (Pierce). The SRA domain from the human UHFR1 protein was purified to homogeneity and crystallized under the conditions described in detail under “Experimental Procedures.” The three-dimensional crystal structure of the SRA domain from human UHFR1 was determined to 1.95 Å resolution (Table 1). As shown in Fig. 1a, its compact architecture is built on an eight-stranded β-barrel core consisting of amino acid sequences conserved in the SRA domain family (supplemental Fig. 1). One side of the β-barrel is curved around helix α1 through interactions with the conserved residues Trp430 and Arg433, whereas the amphipathic helix α2 closes off one end of the barrel by Asn505, Leu508, and Asn511, interacting with the residues in the core of the barrel. Moreover, the C-terminal helix α5 ties together the short N-terminal β1, emphasizing the modular nature of the structure. Note that the loop connecting β4 and α2 is flexible, as residues 484–495 were invisible in the electron density map. Based on SCOP and DALI searches, the structural fold of the SRA domain is distinct topologically from any available protein structures, including the β-barrel OB fold known to interact with nucleic acids (25.Murzin A.G. EMBO J. 1993; 12: 861-867Crossref PubMed Scopus (791) Google Scholar) and the methylated or non-methylated CpG-binding domains (26.Ho K.L. McNae I.W. Schmiedeberg L. Klose R.J. Bird A.P. Walkinshaw M.D. Mol. Cell. 2008; 29: 525-531Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 27.Allen M.D. Grummitt C.G. Hilcenko C. Min S.Y. Tonkin L.M. Johnson C.M. Freund S.M. Bycroft M. Warren A.J. EMBO J. 2006; 25: 4503-4512Crossref PubMed Scopus (127) Google Scholar) (supplemental Fig. 2). The structure exhibits a large, distinct, positively charged patch on the SRA domain surface (Fig. 1b) composed of Arg431, Arg433, Arg443, Lys540, and Arg452. At this positive patch, the electron density map reveals a well ordered sulfate anion (from the crystallization buffer) and a glycerol molecule (from the cryoprotectant solution) that form salt bridges with Arg431 and Arg433, respectively, and a sulfate anion that forms hydrogen bonds with the side chain of Arg443 (Fig. 1a, right panel). We characterized SRA domain/methyl-DNA binding with NMR spectroscopy. As shown in two-dimensional 1H-15N heteronuclear single quantum coherence spectra (Fig. 1c), upon addition of a 13-nucleotide hemimethylated DNA duplex, a large number of protein amide resonances underwent major chemical shift perturbations and conformation exchange-induced line broadening. To identify SRA residues that are important for methyl-DNA recognition, we obtained 90% backbone 1H, 15N, and 13C resonance assignment of the protein with triple-resonance NMR spectra, including residues 484–495 in the β4/α2 loop (supplemental Fig. 3), which are structurally flexible and missing in the electron density map of the crystal structure. Interestingly, the residues that show the most profound chemical shift perturbations or line broadening upon binding to hemimethylated DNA are those located in the β4/α2 loop: Arg431, Phe432, and Arg433 in the β1/α1 loop; Arg443, His445, and Val446 in the α1/β2 loop; and Gly465, Tyr466, Glu467, and Asp469 in the β3/β4 loop (Fig. 1a, right panel). These four loops are spatially clustered at the positive patch of the protein S. J.M. Res. 2003; PubMed Scopus Google Scholar), the that residues the is that many residues in the and β3/β4 loops are in the SRA domain family (supplemental Fig. 1). Using a DNA electrophoretic mobility shift assay used electrophoretic mobility shift we further that the SRA domain a 13-nucleotide or hemimethylated DNA and forms a complex, at with a 13-nucleotide non-methylated DNA (Fig. 2 and 1). To the molecular of methyl-DNA binding by the SRA we performed of Arg433, Arg443, Tyr466, and which are located at the as out by NMR of Arg433 and not a in DNA whereas mutants and in DNA binding with the (Fig. Moreover, mutant the to form a complex with hemimethylated DNA in the Note that the not any of the protein structure as by its NMR to that of the wild-type protein (supplemental Fig. Given that Arg443, Tyr466, and Asp469 are conserved in the SRA domain family (supplemental Fig. we that residues are important for the SRA domain interactions with DNA. To the we determined the dissociation for the binding of the wild-type SRA domain and its mutants to a 5′-fluorescein-labeled 13-nucleotide hemimethylated DNA in a anisotropy binding The the hemimethylated DNA with a Kd of whereas the and mutants a major in binding affinity by and (Fig. The mutant binding to the hemimethylated DNA, with a Kd of as determined by anisotropy with its of DNA binding in the in a in SRA domain binding to hemimethylated DNA to non-methylated DNA of the (supplemental Fig. Moreover, mutant binding to non-methylated DNA was to hemimethylated DNA = 50 a of Asp469 in SRA domain recognition of hemimethylated DNA. In new crystal structure of the human UHRF1 SRA domain to identify key amino acid including Arg443, Tyr466, and at the of the β-barrel that to hemimethylated DNA As residues are conserved the SRA domain we that in this a understanding of the of the SRA domain family in epigenetic gene We the of the NMR at the and the at the Brookhaven National Laboratory X4C and for data We also A. and G. for with

Associations between plants and microorganisms are very complex and are the subject of an increasing number of studies. Here, we specifically address the relationship between poplar and its endophytic bacteria. The role and importance of endophytic bacteria in growth and development of their host plants is still underestimated. However, since many endophytes have a beneficial effect on their host, an improved understanding of the interaction between poplar and its endophytic bacteria has the potential to provide major breakthroughs that will improve the productivity of poplar. Endophytic bacteria can improve plant growth and development in a direct or indirect way. Direct plant growth promoting mechanisms may involve nitrogen fixation, production of plant growth regulators such as auxins, cytokinins and gibberellins, and suppression of stress ethylene synthesis by 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Endophytic bacteria can indirectly benefit the plant by preventing the growth or activity of plant pathogens through competition for space and nutrients, antibiosis, production of hydrolytic enzymes, inhibition of pathogen-produced enzymes or toxins, and through systemic induction of plant defense mechanisms. Examples of applications for custom endophyte-host partnerships include improved productivity and establishment of poplar trees on marginal soils and the phytoremediation of contaminated soils and groundwater. A systems biology approach to understand the synergistic interactions between poplar and its beneficial endophytic bacteria represents an important field of research, which is facilitated by the recent sequencing of the genomes of poplar and several of its endophytic bacteria.

We present the optical design of the Centurion soft X-ray resonant inelastic X-ray scattering (RIXS) spectrometer to be located on the SIX beamline at NSLS-II. The spectrometer is designed to reach a resolving power of 100 000 at 1000 eV at its best resolution. It is also designed to have continuously variable 2θ motion over a range of 112° using a custom triple rotating flange. We have analyzed several possible spectrometer designs capable of reaching the target resolution. After careful analysis, we have adopted a Hettrick-Underwood spectrometer design, with an additional plane mirror to maintain a fixed direction for the outgoing beam. The spectrometer can cancel defocus and coma aberrations at all energies, has an erect focal plane, and minimizes mechanical motions of the detector. When the beamline resolution is accounted for, the net spectral resolution will be 14 meV at 1000 eV. This will open up many low energy excitations to study and will expand greatly the power of soft X-ray RIXS.