Anomalous Diffraction Experiments Research Articles

Anomalous x-ray diffraction studies of Sr2+ hydration in aqueous solution

Anomalous x-ray diffraction experiments were carried out at two energies below the K edge of strontium on a 3.5 molal solution of strontium chloride in water. The first order difference method was applied to the corrected and normalized data sets, and a difference function for the Sr2+ coordination obtained. Fourier transformation of this function gave a modified total radial distribution function for Sr2+, which when analyzed showed that there are eight to nine nearest neighbor water molecules sited at 2.67 Å from the Sr2+ ion. A second coordination shell is also apparent in the range 4.5⩽r(Å)⩽5.5 which can accommodate Cl− counterions and a second shell of water molecules. The results obtained for the distance between Sr2+ and O are in excellent agreement with those obtained from standard x-ray diffraction, extended x-ray absorption fine structure, and neutron diffraction isotopic substitution. The hydration structure obtained for Sr2+ is compared with the structure of two other ions in the alkaline earth series (Mg2+ and Ca2+).

Anomalous x-ray diffraction on InAs/GaAs quantum dot systems

Free-standing InAs quantum dots on a GaAs (001) substrate have been investigated using grazing incidence x-ray diffraction. To suppress the strong scattering contribution from the GaAs substrate, we performed anomalous diffraction experiments at the superstructure (200) reflection, showing that the relative intensities from the dots and the substrate undergo a significant change with the x-ray energy below and above the As K edge. Since the signal from the substrate material can essentially be suppressed, this method is ideally suited for the investigation of strain, shape, and interdiffusion of buried quantum dots and quantum dots embedded in heteroepitaxial multilayers. In addition, we show that it can be used as a tool for studying wetting layers.

The Crystal Structure of the Olfactory Marker Protein at 2.3 Å Resolution

Olfactory marker protein (OMP) is a highly expressed and phylogenetically conserved cytoplasmic protein of unknown function found almost exclusively in mature olfactory sensory neurons. Electrophysiological studies of olfactory epithelia in OMP knock-out mice show strongly retarded recovery following odorant stimulation leading to an impaired response to pulsed odor stimulation. Although these studies show that OMP is a modulator of the olfactory signal-transduction cascade, its biochemical role is not established. In order to facilitate further studies on the molecular function of OMP, its crystal structure has been determined at 2.3 Å resolution using multiwavelength anomalous diffraction experiments on selenium-labeled protein. OMP is observed to form a modified β-clamshell structure with eight antiparallel β-strands. While OMP has no significant sequence homology to proteins of known structure, it has a similar fold to a domain found in a variety of existing structures, including in a large family of viral capsid proteins. The surface of OMP is mostly convex and lacking obvious small molecule binding sites, suggesting that it is more likely to be involved in modulating protein–protein interaction than in interacting with small molecule ligands. Three highly conserved regions have been identified as leading candidates for protein–protein interaction sites in OMP. One of these sites represents a loop known to mediate ligand interactions in the structurally homologous EphB2 receptor ligand-binding domain. This site is partially buried in the crystal structure but fully exposed in the NMR solution structure of OMP due to a change in the orientation of an α-helix that projects outward from the structurally invariant β-clamshell core. Gating of this conformational change by molecular interactions in the signal-transduction cascade could be used to control access to OMP's equivalent of the EphB2 ligand-interaction loop, thereby allowing OMP to function as a molecular switch.

Crystal Structure of the Autocatalytic Initiator of Glycogen Biosynthesis, Glycogenin

Glycogen is an important storage reserve of glucose present in many organisms, from bacteria to humans. Its biosynthesis is initiated by a specialized protein, glycogenin, which has the unusual property of transferring glucose from UDP–glucose to form an oligosaccharide covalently attached to itself at Tyr194. Glycogen synthase and the branching enzyme complete the synthesis of the polysaccharide. The structure of glycogenin was solved in two different crystal forms. Tetragonal crystals contained a pentamer of dimers in the asymmetric unit arranged in an improper non-crystallographic 10-fold relationship, and orthorhombic crystals contained a monomer in the asymmetric unit that is arranged about a 2-fold crystallographic axis to form a dimer. The structure was first solved to 3.4 Å using the tetragonal crystal form and a three-wavelength Se–Met multi-wavelength anomalous diffraction (MAD) experiment. Subsequently, an apo-enzyme structure and a complex between glycogenin and UDP–glucose/Mn 2+ were solved by molecular replacement to 1.9 Å using the orthorhombic crystal form. Glycogenin contains a conserved D×D motif and an N-terminal β-α-β Rossmann-like fold that are common to the nucleotide-binding domains of most glycosyltransferases. Although sequence identity amongst glycosyltransferases is minimal, the overall folds are similar. In all of these enzymes, the D×D motif is essential for coordination of the catalytic divalent cation, most commonly Mn 2+. We propose a mechanism in which the Mn 2+ that associates with the UDP–glucose molecule functions as a Lewis acid to stabilize the leaving group UDP and to facilitate the transfer of the glucose moiety to an intermediate nucleophilic acceptor in the enzyme active site, most likely Asp162. Following transient transfer to Asp162, the glucose moiety is then delivered to the final acceptor, either directly to Tyr194 or to glucose residues already attached to Tyr194. The positioning of the bound UDP–glucose far from Tyr194 in the glycogenin structure raises questions as to the mechanism for the attachment of the first glucose residues. Possibly the initial glucosylation is via inter-dimeric catalysis with an intra-molecular mechanism employed later in oligosaccharide synthesis.

Design of a 5-Station Macromolecular Crystallography Beamline at MAX-Lab

A beamline for macromolecular crystallography is under construction at the Swedish synchrotron light source MAX-lab at Lund University in a collaborative effort between Denmark and Sweden. Of the 7 mrad horizontal wiggler fan emitted from the new superconducting multipole wiggler, the central 2 mrad will be used and split in three parts. The central 1 mrad will be used for a tunable station optimised for multi-wavelength anomalous diffraction experiments and on each side of the central fan there will be two fixed wavelength stations using different energies of the same part of the beam. These in total five stations can be used simultaneously and independently for collecting diffraction data.

FIP: a highly automated beamline for multiwavelength anomalous diffraction experiments.

FIP is a French Collaborating Research Group (CRG) beamline at the European Synchrotron Radiation Facility (ESRF) dedicated exclusively to crystallography of biological macromolecules, with a special emphasis on multiwavelength anomalous diffraction data collection in the 0.7-1.81 A wavelength range. The optics, consisting of long cylindrical grazing-angle mirrors associated with a cryocooled double-crystal monochromator, delivers an optimal beam in the corresponding energy range. The high level of automation, which includes automated crystal centring, automated data-collection management and data processing, makes the use of this beamline very easy. This is illustrated by the large number of challenging structures that have been solved since 1999.

Crystal structure of the Escherichia coli SbmC protein that protects cells from the DNA replication inhibitor microcin B17.

Crystal structure of the Escherichia coli SbmC protein that protects cells from the DNA replication inhibitor microcin B17.

X-ray crystallographic studies of serotonin N-acetyltransferase catalysis and inhibition

The structure of serotonin N-acetyltransferase (also known as arylalkylamine N-acetyltransferase; AANAT) bound to a potent bisubstrate analog inhibitor has been determined at 2.0 Å resolution using a two-edge (Se, Br) multiwavelength anomalous diffraction (MAD) experiment. This acetyl-CoA dependent enzyme is a member of the GCN5-related family of N-acetyltransferases (GNATs), which share four conserved sequence motifs (A-D). In serotonin N-acetyltransferase, motif A adopts an α/β conformation characteristic of the phylogenetically invariant cofactor binding site seen in all previously characterized GNATs. Motif B displays a significantly lower level of conservation among family members, giving rise to a novel α/β structure for the serotonin binding slot. Utilization of a brominated CoA- S-acetyl-tryptamine-bisubstrate analog inhibitor and the MAD method permitted conclusive identification of two radically different conformations for the tryptamine moiety in the catalytic site ( cis and trans). A second high-resolution X-ray structure of the enzyme bound to a bisubstrate analog inhibitor, with a longer tether between the acetyl-CoA and tryptamine moieties, demonstrates only the trans conformation. Given a previous proposal that AANAT can catalyze an alkyltransferase reaction in a conformationally altered active site relative to its acetyltransferase activity, it is possible that the two conformations of the bisubstrate analog observed crystallographically correspond to these alternative reaction pathways. Our findings may ultimately lead to the design of analogs with improved AANAT inhibitory properties for in vivo applications.

Overview of the tunable beamlines for protein crystallography at the EMBL Hamburg Outstation; an analysis of current and future usage and developments

The EMBL Hamburg Outstation currently operates two tunable protein crystallography beamlines suitable for single and multiple anomalous diffraction (SAD/MAD) experiments. The first beamline, designated X31, is located on a bending magnet of the DORIS III storage ring whereas the second beamline, BW7A, is positioned at a multipole wiggler at the same storage ring. X31 is equipped with an energy stabilization device to ensure constant wavelength during longer data-collection periods. The in-house built crystallographic end-station is now equipped with a Mar345 imaging-plate scanner as a detector. The wiggler beamline BW7A features a novel sagitally focusing monochromator. The end-station used here has also been developed and built in-house. The beamline is currently operated with a Mar 165 CCD detector. In this paper the hardware and software developments of the last years will be summarized and the outlook for substantial upgrades will be given. The future plans include the design and construction of a third tunable beamline, designated X12, for protein crystallography. The development of automated beamlines for protein crystallography is of particular importance with respect to structural genomics initiatives. The analysis of the projects of the last years shows the wide range of anomalous scatterer used on the tunable beamlines thus demonstrating the need of a wide range of accessible energies and fast and reliable energy changes.

A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase

EST2 is a novel thermophilic carboxylesterase, isolated and cloned from Alicyclobacillus (formerly Bacillus) acidocaldarius, which optimally hydrolyses esters with acyl chain lengths of six to eight carbon atoms at 70°C. On the basis of the amino acid sequence homology, it has been classified as a member of the mammalian hormone-sensitive lipase (HSL) subfamily.The crystal structure of EST2, complexed with a sulphonyl derivative, has been determined at 2.6 Å resolution by a multiple wavelength anomalous diffraction experiment on a seleno-methionine derivative. EST2 presents a canonical α/β hydrolase core, shielded at the C-terminal side by a cap region built up of five helices. It contains the lipase-like catalytic triad, Ser155, His282 and Asp252, whereby the nucleophile is covalently modified. This allows an unambiguous view of the putative active site of EST2, detecting the oxyanion hole, in whose formation the amino acid sequence motif His81-Gly82-Gly83-Gly84 is involved, and the hydrophobic binding pocket for the acyl chain. The structural model here reported provides the first example of a transition state analogue of an esterase/lipase belonging to the HSL group, thus affording useful information for the design of medical inhibitors. Moreover, as the first X-ray structure of a thermophilic carboxylesterase, the comparison with its mesophilic homologue, the Brefeldin A esterase (BFAE) from Bacillus subtilis, allows the identification of putative determinants of thermal stability.

The challenge of anomalous diffraction experiments at the MVedge (3.5 Å) of uranium

The challenge of anomalous diffraction experiments at the M_Vedge (3.5 Å) of uranium

Local structure of In0.5Ga0.5As from joint high-resolution and differential pair distribution function analysis

High resolution total and indium differential atomic pair distribution functions (PDFs) for In0.5Ga0.5As alloys have been obtained by high energy and anomalous x-ray diffraction experiments, respectively. The first peak in the total PDF is resolved as a doublet due to the presence of two distinct bond lengths, In–As and Ga–As. The In differential PDF, which involves only atomic pairs containing In, yields chemical specific information and helps ease the structure data interpretation. Both PDFs have been fit with structure models and the way in that the underlying cubic zinc-blende lattice of In0.5Ga0.5As semiconductor alloy distorts locally to accommodate the distinct In–As and Ga–As bond lengths present has been quantified.

Screening orthologs as an important variable in crystallization: preliminary X-ray diffraction studies of the tRNA-modifying enzyme S-adenosyl-methionine:tRNA ribosyl transferase/isomerase.

The genes encoding the tRNA-modifying enzyme S-adenosylmethionine:tRNA ribosyl transferase/isomerase (QueA) from 12 eubacterial sources were overexpressed in Escherichia coli and the resulting products were purified to homogeneity and subjected to crystallization trials. Using the hanging-drop vapour-diffusion method, crystals suitable for X-ray diffraction experiments were only obtained for the queA gene product from Bacillus subtilis. The crystals belong to the space group P422, with unit-cell parameters a = b = 100.7, c = 150.9 A. Using highly focused synchrotron radiation from the EMBL/ESRF beamline ID13 (Grenoble, France), diffraction to at least 3.2 A could be achieved. A selenomethionyl derivative of the protein was prepared and crystallized for future multiwavelength anomalous diffraction (MAD) experiments.

A secondary graphite crystal spectrometer for anomalous X-ray diffraction experiments

Abstract A new design for implementation of anomalous X-ray diffraction experiments is proposed. The exploitation of a graphite crystal spectrometer with good energy resolution in combination with an acceptable counting rate opens new possibilities to carry out AWAXS experiments and calculate partial structure functions. The proof of this measurement principle is demonstrated by presentation of the partial structure factor and radial distribution function for rubidium germanate glasses around the germanium component.

A MAD experiment performed at the white line of the iridium LIII absorption edge in lysozyme.

A multiple-wavelength anomalous diffraction (MAD) experiment was performed on an iridium derivative of hen egg-white lysozyme. Diffraction data were measured at five wavelengths on the X31 EMBL beamline on the DORIS III storage ring and at two further wavelengths on the X11 beamline. Four iridium-binding sites were located from the dispersive anomalous differences between two wavelengths at the rising and falling inflection points of the Ir LIII-edge white line using direct methods. All other attempts to determine the heavy-atom positions failed. The results demonstrate an experimental method whereby systematic error in MAD data due to sample absorption can be reduced where a white line is present in the absorption spectrum of a heavy atom.

Sequence-specific recognition of the internalization motif of the Alzheimer's amyloid precursor protein by the X11 PTB domain.

The crystal structure of the phosphotyrosine-binding domain (PTB) of the X11 protein has been determined, in complex with unphosphorylated peptides corresponding to a region of beta-amyloid precursor protein (betaAPP) that is required for receptor internalization. The mode of binding to X11 of the unphosphorylated peptides, which contain an NPxY motif, resembles that of phosphorylated peptides bound to the Shc and IRS-1 PTB domains. Eight peptide residues make specific contacts with the X11 PTB domain, and they collectively achieve high affinity (KD = 0.32 microM) and specificity. These results suggest that, in contrast to the SH2 domains, the PTB domains are primarily peptide-binding domains that have, in some cases, acquired specificity for phosphorylated tyrosines.

The structure of ribosomal protein S7 at 1.9 A resolution reveals a beta-hairpin motif that binds double-stranded nucleic acids.

Ribosomal protein S7, a crucial RNA-binding component of the ribosome, is one of two proteins that initiates assembly of the 30S ribosomal subunit. It is required for proper folding of a large 3' domain of 16S ribosomal RNA. S7 regulates its own synthesis by binding to its own mRNA. This ability of S7 to bind both messenger and ribosomal RNAs makes determination of its mode of RNA recognition particularly interesting. The crystal structure of S7 from Thermus thermophilus was determined by a two-wavelength anomalous diffraction experiment using the LIII edge of mercury. The S7 structure consists of a bundle of six helices and an extended beta hairpin between helices 3 and 4, with two or more RNA-binding sites on its surface. The hairpin, along with portions of helices 1, 4 and 6, forms a large, positively charged, concave surface that has the appropriate curvature and dimensions to bind double-stranded RNA. A second putative RNA-binding site comprises parts of loop 2 and the helix 4-loop 5 turn. Structural similarity between S7 and the IHF/HU family of proteins strongly suggests that the beta hairpin of S7 binds to a groove of double-stranded RNA. The beta hairpin of S7 is also similar to those from other nucleic acid binding proteins, such as ribosomal protein L14 and BIV Tat, suggesting that it belongs to an extended family of such motifs, all of which bind to a groove of double-stranded nucleic acid. The residues in S7 loop 2 that belong to the second putative RNA-binding site may have a role analogous to the N-terminal residues of IHF/HU which grip an unbent portion of double helix.

An Incident-Beam Monitor for Use in Protein Crystallography at a Synchrotron Source

A compact incident-beam monitor has been developed for use in synchrotron protein crystallography at the National Synchrotron Light Source beamline X8C. Incident-beam intensity is monitored by measurement of the radiation scattered from a thin polymer film into a PIN diode. For improved statistical accuracy, the scattering film can be replaced by a thin metal foil. Fluorescent radiation emitted by the metal foil increases the beam-monitoring signal up to 50 times relative to that produced by scattering alone. With this design, the forward-scattered radiation is restricted to an area not larger than the beam stop, minimizing excessive background radiation. Support materials have been optimized so that no unwanted edge effects are present between 6 and 19.5 keV, making the detector useful for monitoring incident-beam intensities over the wide range of absorption edges often associated with multiwavelength anomalous diffraction (MAD) experiments. Accurate incident-beam monitoring also simplifies optimization of the X-ray beam through the diffractometer collimation after each new electron orbit is established in the synchrotron.

Determining Phases and Anomalous-Scattering Models from the Multiwavelength Anomalous Diffraction of Native Protein Metal Clusters. Improved MAD Phase Error Estimates and Anomalous-Scatterer Positions

A strategy is presented for refining anomalous scattering models and calculating protein phases directly from the Bijvoet and dispersive differences of a macromolecular multiwavelength anomalous diffraction (MAD) experiment. This procedure, incorporated in the program MADPHSREF, is especially amenable for exploiting the weak perturbations to normal scattering produced by inner-shell electronic transitions of asymmetric metal and protein ligand assemblies. The protocol accounts for more than one type of anomalous scatterer, incorporates stereochemical restraints, treats the data in local scaling groups, and partly compensates for correlated errors. Approximating maximum likelihood by averaging observation variances and covariances over all values of phase considerably improved error estimation. Probabilistic rejection of aberrant observations, re-evaluated before each refinement cycle, improved refinement convergence and accuracy compared with other less flexible rejection criteria. MADPHSREF allows the facile combination of MAD phase information with phase information from other sources. For the suifite reductase hemoprotein (SiRHP), relative weights for MAD and multiple isomorphous replacement (MIR) phases were determined by matching histograms of electron density. Accurate metal-cluster geometries and the associated errors in atomic positions can be determined from refinement against anomalous differences using normal scattering phases from a refined structure. When applied to MAD data collected on SiRHP, these methods confirmed the Fe(4)S(4) cluster asymmetry initially observed in the refined 1.6 A resolution structure and resulted in a MAD-phased, experimental, electron-density map that is of better quality than the combined MAD/MIR map originally used to determine the structure.

A high-power and high-flux X-ray wiggler station at the Cornell high energy synchrotron source

The F2 wiggler station at The Cornell High Energy Synchrotron Source has been in operation since August of 1990. This versatile stationis designed to accommodate a wide variety of state-of-the-art synchrotron experiments. The station can produce tunable doubly-focused monochromatic radiation as well as unfocused white beam. Macromolecular crystallography experiments using multiple wavelength anomalous diffraction (MAD) and high resolution diffraction experiments have taken advantage of the high flux, high energy resolution, and the tunability of the station in the monochromatic mode; nano-eV spectroscopy experiments using nuclear resonant scattering have utilized the high flux available at a particular energy; and high heat load tests of X-ray optics have used the full power of the white beam. In this article we present several key elements of the station design and review the X-ray beam characteristics as they pertain to the scientific work done at F2 during the past few years.