Insights from soft X-rays: the chlorine and sulfur sub-structures of a CK2α/DRB complex

The diffraction pattern of a protein crystal is normally a product of the interference of electromagnetic waves scattered by electrons of the crystalline sample. The diffraction pattern undergoes systematic changes in case additionally X-ray absorption occurs, meaning if the wavelength of the primary X-ray beam is relatively close to the absorption edge of selected elements of the sample. The resulting effects are summarized as "anomalous dispersion" and can be always observed with "soft" X-rays (wavelength around 2 A) since they match the absorption edges of sulfur and chlorine. A particularly useful application of this phenomenon is the experimental detection of the sub-structures of the anomalous scatterers in protein crystals. We demonstrate this here with a crystal of a C-terminally truncated variant of human CK2alpha to which two molecules of the inhibitor 5,6-dichloro-1-beta-D: -ribo-furanosyl-benzimidazole (DRB) are bound. The structure of this co-crystal has been solved recently. For this study we measured an additional diffraction data set at a wavelength of 2 A which showed strong anomalous dispersion effects. On the basis of these effects we detected all sulfur atoms of the protein, the two liganded DRB molecules and a total of 16 additional chloride ions some of them emerging at positions filled with water molecules in previous structure determinations. A number of chloride ions are bound to structural and functional important locations fitting to the constitutive activity and the acidophilic substrate specificity of the enzyme.     

Crystallization and preliminary X-ray diffraction studies of the human adenovirus serotype 2 proteinase with peptide cofactor.

Recombinant human adenovirus serotype 2 proteinase (both native and selenomethionine-substituted) has been crystallized in the presence of the serotype 12, 11-residue peptide cofactor. The crystals (space group P3(1)21 or P3(2)21, one molecule per asymmetric unit, a = b = 41.3 angstrum, c = 197.0 angstrum) grew in solutions containing 20-40% 2-methyl-2,4-pentanediol (MPD), 0.1-0.2 M sodium citrate, and 0.1 M sodium HEPES, pH 5.0-7.5. Diffraction data (84% complete to 2.2 angstrum resolution with Rmerge of 0.0335) have been measured from cryopreserved native enzyme crystals with the Argonne blue (1,024 x 1,024 pixel array) charge-coupled device detector at beamline X8C at the National Synchrotron Light Source (operated by Argonne National Laboratory's Structural Biology Center). Additionally, diffraction data from selenomethionine-substituted proteinase, 65% complete to 2.0 angstrum resolution with Rmerge values ranging 0.05-0.07, have been collected at three X-ray energies at and near the selenium absorption edge. We have determined three of the six selenium sites and are initiating a structure solution by the method of multiwavelength anomalous diffraction phasing.     

Structure and composition of myelinated axons: a multimodal synchrotron spectro-microscopy study

We report elemental mappings on the sub-cellular level of myelinated sciatic neurons isolated from wild type mice, with high spatial resolution. The distribution of P, S, Cl, Na, K, Fe, Mn, Cu was imaged in freeze-dried as well as cryo-preserved specimen, using the recently developed cryogenic sample environment at beamline ID21 at the European Synchrotron Radiation Facility (ESRF). In addition, synchrotron radiation based Fourier transform infrared (FTIR) spectromicroscopy was used as a chemically sensitive imaging method. Finally single fiber diffraction in highly focused hard X-ray beams, and soft X-ray microscopy and tomography in absorption contrast are demonstrated as novel techniques for the study of single nerve fibers.     

Crystal and molecular structure and absolute configuration of lincomycin hydrochloride monohydrate

Lincomycin is a broad-spectrum antibiotic synthesized by Streptomyces lincolnensis that is particularly active against Gram-positive bacteria. It is widely used in human and veterinary applications. The crystal structure of lincomycin has been undertaken with a view to obtain the conformational and structural features of the drug in order to afford a comparison of its structural features with other aminoglycoside antibiotics. We report here the details of its structural and conformational features as determined by single-crystal X-ray crystallography. Crystals of lincomycin hydrochloride are orthorhombic, space group P2(1)2(1)2, with the cell dimensions a=18.5294(3) Angstroms, b=20.5980(4) Angstroms, c=6.17380(10) Angstroms, V=2356.35(7) Angstroms3. The structure was solved using X-ray diffraction data and refined to a final R-value of 0.0391 for 2321 reflections (I > or = 2sigma). The absolute configuration was established using the anomalous dispersion of the sulfur and chlorine atoms in the structure. The molecule consists of an amino acid linked by an amide group to a monosaccharide of galactose stereochemistry. A network of hydrogen-bonds stabilizes the crystal structure.     

X-ray absorption near edge structures of DNA or its components around the oxygen K-shell edge

The initial process of radiation damage in DNA was investigated by measuring the X-ray absorption near edge structures (XANES) within the energy region around the oxygen K-shell absorption edge for DNA, cytosine and 2-deoxy-d-ribose. Irradiation and XANES experiments were performed with the BL23SU soft X-ray beamline, using synchrotron radiation from the 8 GeV electron storage ring at SPring-8. Samples were mounted on gold-coated plates in a vacuum chamber. The XANES spectra were obtained by measuring the photoelectron current of the samples. 2-Deoxy-d-ribose was exposed to X rays at the absorption peak corresponding to the oxygen (O) 1s-->sigma* transition energy (538 eV); the XANES spectra were obtained after each irradiation. DNA and cytosine, possessing characteristic XANES spectra, both had two major energy bands corresponding to the O 1s-->pi* and 1s-->sigma* transitions. Two new peaks appeared and gradually increased in the XANES spectra of 2-deoxy-d-ribose during irradiation. These results suggest that C-O bonds in 2-deoxy-d-ribose are transformed to C=O bonds by O 1s-->sigma* transition, suggesting that the molecules undergo chemical changes into carbonyl-containing compounds.     

The beamlines of ELETTRA and their application to structural biology

Protein crystallographers are nowadays regular users of synchrotron radiation (SR) facilities for several applications. The goal of majority of users is simply to extract more accurate, higher resolution data from existing crystals; they use monochromatic radiation and the rotation method, in order to get a complete survey of the reciprocal space in a short time. In fact the brilliance of SR is essential, due to the weak scattering power of the samples, and because of their sensibility to radiation damage. Over the last few years, however, a general increase of interest for measurements at multiple wavelengths, which exploit the anomalous dispersion for the phase problem (multiwavelength anomalous diffraction — MAD), has generated the need of intense tuneable sources. For these applications, the emphasis is on accurate measurements of the small differences between the intensities of Bragg reflections at various energies across the absorption edge of an element present in the sample. The macromolecular diffraction beamline at ELETTRA, which is now running routinely since spring 1995, has been designed to provide a high flux — highly collimated tuneable X-rays source in the spectral range between 4 and 25 keV. The radiation source is the 57-pole wiggler, which delivers a very intense radiation up to 25 keV, and is shared and used simultaneously with the small angle X-ray scattering (SAXS) beamline. The front-end filter system has a cut-off energy at about 4 keV. The beamline optics consists of a pseudo-channel-cut double-crystal monochromator followed by a double focusing toroidal mirror. The tunability and the stability of the monochromator allows the user to perform MAD experiments, and for this purpose, a fluorescence probe for the exact calibration of the absorption edge is available on-line. The experimental station is based on an imaging plate area detector from MarResearch, with a sensible area of 345 mm in diameter. A cooled N₂-stream is available to cool the sample crystal in order to reduce the radiation damage. SAXS is an experimental technique used to derive structural information about supra-molecular assemblies, amorphous materials and partly ordered systems (e.g. size and shape of large molecules). The high-flux SAXS beamline at ELETTRA is mainly intended for time-resolved studies on fast structural transitions in the sub-millisecond time region in solutions and in partly ordered systems, triggered by external or process parameters, with a SAXS resolution between 10 and 1400 Å in real space. The source is the already mentioned 57-pole and the SAXS beamline accepts three discrete energies of its spectrum, namely 5.4, 8 and 16 keV. The beamline optics consists of a flat double-crystal monochromator and a double focusing toroidal mirror. A multi-purpose sample stage, movable along an optical table in order to optimise the sample to detect distance, allows to perform fast time-resolved relaxation studies based on temperature- or pressure-jumps as well as stopped flow experiments. Moreover, the users have option to install their own specialised sample surrounding equipment. The optimisation of the beamline with respect to high-flux and consequently high-flux density, allows to perform the following experiments: low contrast solution scattering, grazing incidence surface diffraction, micro-spot scanning, X-ray fluorescence analysis, time-resolved studies 11 s, simultaneous small- and wide-angle measurements on gels, liquid crystals, biopolymers, amorphous materials, muscles.This revised version was published online in October 2005 with corrections to the Cover Date.     

X-ray structure determination of a vanadium-dependent haloperoxidase from Ascophyllum nodosum at 2.0 A resolution.

The homo-dimeric structure of a vanadium-dependent haloperoxidase (V-BPO) from the brown alga Ascophyllum nodosum (EC 1.1.11.X) has been solved by single isomorphous replacement anomalous scattering (SIRAS) X-ray crystallography at 2.0 A resolution (PDB accession code 1QI9), using two heavy-atom datasets of a tungstate derivative measured at two different wavelengths. The protein sequence (SwissProt entry code P81701) of V-BPO was established by combining results from protein and DNA sequencing, and electron density interpretation. The enzyme has nearly an all-helical structure, with two four-helix bundles and only three small beta-sheets. The holoenzyme contains trigonal-bipyramidal coordinated vanadium atoms at its two active centres. Structural similarity to the only other structurally characterized vanadium-dependent chloroperoxidase (V-CPO) from Curvularia inaequalis exists in the vicinity of the active site and to a lesser extent in the central four-helix bundle. Despite the low sequence and structural similarity between V-BPO and V-CPO, the vanadium binding centres are highly conserved on the N-terminal side of an alpha-helix and include the proposed catalytic histidine residue (His418(V-BPO)/His404(V-CPO)). The V-BPO structure contains, in addition, a second histidine near the active site (His411(V-BPO)), which can alter the redox potential of the catalytically active VO2-O2 species by protonation/deprotonation reactions. Specific binding sites for the organic substrates, like indoles and monochlordimedone, or for halide ions are not visible in the V-BPO structure. A reaction mechanism for the enzymatic oxidation of halides is discussed, based on the present structural, spectroscopic and biochemical knowledge of vanadium-dependent haloperoxidases, explaining the observed enzymatic differences between both enzymes.     

Sulfur distribution in bacteriorhodopsin from multiple wavelength anomalous diffraction near the sulfur K-edge with synchrotron x-ray radiation.

Bacteriorhodopsin contains nine sulfur atoms from the nine methionine residues. The distribution of these sulfur atoms in the projected density map was determined from x-ray diffraction experiments using multiple wavelength anomalous diffraction (MAD) at the sulfur K-edge (5.02 A) with synchrotron radiation. The experiments were performed with uniaxial samples of oriented purple membranes at room temperature and 86% relative humidity. For such samples only the real part f' (lambda) of the resonant scattering amplitude of sulfur contributes to the observed scattering intensity. The sulfur density was determined from the difference in diffraction intensities detected at two wavelengths near the sulfur K-edge that were approximately 0.004 A apart. The measured change in f' between these two wavelengths corresponds to 6 electron units. This shows that large anomalous dispersion effects occur near the sulfur K-edge. The in-plane positions of the sulfur atoms of Met32, Met56, and Met209 were determined unambiguously. The difference density from Met20, Met60, Met118, and Met145 is concentrated in the interior of the seven alpha-helical bundle, overlaps strongly in the projected density map, and cannot be resolved at the resolution of these experiments (8.2 A). This method of localizing individual sulfur atoms can be applied to other two-dimensional protein crystals and is promising in conjunction with the site-directed introduction of sulfur atoms by the use of cysteine mutants.     

Trichromatic concept optimizes MAD experiments in synchrotron X-ray crystallography

The trichromatic concept is a new synchrotron beamline design that optimizes MAD experiments by reducing systematic experimental errors with three-colored and coaxial synchrotron X-ray beams produced by a tandem vertical undulator and trichromator. The concept enables rapid and flexible switching of three defined wavelengths, and extends the flexibility of experimental design for MAD data collection. Thus, we can collect MAD data taking into account time series effects such as radiation damage. The data based on the trichromatic concept gave a better quality electron density map than data collected by conventional methods. It was also revealed that multicolor diffraction using dichromatic or trichromatic X-ray beams is effective in rapid MAD data collection.     

Structure and growth of ultrasmall protein microcrystals by synchrotron radiation: I. microGISAXS and microdiffraction of P450scc

Ultrasmall P450scc cytochrome microcrystals are grown by classical hanging vapor diffusion and by its modification using homologous protein thin-film template displaying a long-range order. The nucleation and growth mechanisms of P450scc microcrystals are studied at the thin cytochrome film surface by a new microbeam grazing incidence small angle X-ray scattering (microGISAXS) technique developed at the microfocus beamline of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. P450scc cytochrome crystals of about 5 microm are also investigated by synchrotron radiation diffraction in order to attempt a preliminary analysis of the atomic structure of this unique protein system yet unsolved.     

Crystal structure study of Opsanus tau parvalbumin by multiwavelength anomalous diffraction

The crystal structure of a small calcium-binding protein, the parvalbumin IIIf from Opsanus tau in which Tb was substituted for Ca, has been analysed by multiwavelength anomalous diffraction. Data at a resolution of 2.3 A were collected at three wavelengths near the L3 absorption edge of Tb (1.645-1.650 A), using the synchrotron radiation emitted by a storage ring and a multiwire proportional counter. The phases of the reflections were determined from this single derivative, without native data. Prior to any refinement, the resulting electron density map shows a good agreement with the model of the homologous carp parvalbumin in regions of identical amino-acid sequence.     

Conformation and absolute configuration of 2-naphthylalkylsulfoxides by combined use of dynamic NMR, ECD spectroscopy, DFT computations, and X-ray diffraction

The E/Z conformer ratios of five 2-naphtylalkylsulfoxides were determined by means of dynamic NMR spectroscopy at very low temperatures and this information was used to predict, by means of DFT calculations, the ECD spectra of the R and S enantiomers. The latter were separated by enantioselective HPLC technique and the comparison of theoretical and experimental ECD spectra allowed the absolute configurations to be determined. In the case of 2-naphtyl tert-butylsulfoxide (1), the assignment was independently confirmed by anomalous dispersion using single crystal X-ray crystallography.     

Crystallization and MAD data collection of high-molecular weight cytochrome c from Desulfovibrio vulgaris Miyazaki F

Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.     

X-ray diffraction from a single layer of purple membrane at the air/water interface

X-ray diffraction patterns have been recorded from a single layer of purple membrane ( approximately 50 A thickness) at the air/water interface in a Langmuir trough. Grazing-incidence X-ray diffraction is demonstrated to be a promising method for obtaining structural information on membrane proteins under physiological conditions. The method is so sensitive that diffraction can be measured from samples with only 10(13) protein molecules in the beam. Diffraction from hexagonal crystals of purple membrane with a lattice constant of 61. 3 A was observed up to the order {h,k}={4,3}, corresponding to a resolution of approximately 9 A. The work reported here is a first step towards a new way of protein crystallography using grazing-incidence X-ray diffraction at the air/water interface.     

Substrate recognition and processing by a Walker B mutant of the human mitochondrial AAA+ protein CLPX

Soft X-ray cryo-microscopy/tomography of vitreous samples is becoming a valuable tool in structural cell biology. Within the 'water-window' wavelength region (2.34-4.37nm), it provides absorption contrast images with high signal to noise ratio and resolution of a few tens of nanometer. Soft X-rays with wavelengths close to the K-absorption edge of oxygen penetrate biological samples with thicknesses in the micrometer range. Here, we report on the application of a recently established extension of the transmission soft X-ray cryo-microscope (HZB TXM) at the beamline U41-XM of the BESSY II electron storage ring by an in-column epi-fluorescence and reflected light cryo-microscope. We demonstrate the new capability for correlative fluorescence and soft X-ray cryo-microscopy/tomography of this instrument along a typical life science experimental approach - the correlation of a fluorophore-tagged protein (pUL34-GFP of pseudorabies virus, PrV, the nuclear membrane-anchored component of the nuclear egress complex of the Herpesviridae which interacts with viral pUL31) in PrV pUL34-GFP/pUL31 coexpressing mammalian cells, with virus-induced vesicular structures in the nucleus, expanding the nucleoplasmic reticulum. Taken together, our results demonstrate new possibilities to study the role of specific proteins in substructures of adherent cells, especially of the nucleus in toto, accessible to electron microscopy in thinned samples only.     

Crystallization and preliminary X-ray diffraction analysis of monoprenylated Rab7 GTPase in complex with Rab escort protein 1

Posttranslational geranylgeranylation of Rab GTPases is catalyzed by Rab geranylgeranyltransferase (RabGGTase), which consists of a catalytic alpha/beta heterodimer and an accessory Rab escort protein (REP). REP functions as a molecular chaperone that presents Rab proteins to the RabGGTase and after prenylation delivers them to their target membrane. Mutations in the REP-1 gene in humans lead to an X-chromosome-linked defect known as choroideremia, a progressive disease that inevitably culminates in complete blindness. Here we report in vitro assembly, purification, and crystallization of the monoprenylated Rab7GDP:REP-1 complex. X-Ray diffraction data for the REP-1:Rab7 complex were collected to 2.2-A resolution at the ESRF. The crystals belong to the orthorhombic space group P2(1)2(1)2 with unit-cell parameters a=64.3A, b=105.3A, c=132.6A. Preliminary structural analysis revealed the presence of one complex in the asymmetric unit. To understand the conformational changes in Rab protein on complex formation we also crystallized the GDP-bound form of Rab7 that diffracted to at least 1.8A on the in-house X-ray source.     

Crystallization and preliminary X-ray diffraction analysis of the Rab escort protein-1 in complex with Rab geranylgeranyltransferase

Posttranslational prenylation of proteins is a widespread phenomenon and the majority of prenylated proteins are geranylgeranylated members of the Rab GTPase family. Geranylgeranylation is catalyzed by Rab geranylgeranyltransferase (RabGGTase) and is critical for the ability of Rab protein to mediate vesicular docking and fusion of various intracellular vesicles. RabGGTase consists of a catalytic alpha/beta heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane. Mutations in the REP-1 gene in humans lead to an X-chromosome-linked defect known as choroideremia--a debilitating disease that inevitably culminates in complete blindness. Here we report in vitro assembly and purification of the stoichiometric ternary complex of RabGGTase with REP-1 stabilized by a hydrolysis-resistant phosphoisoprenoid analog--farnesyl phosphonyl(methyl)phoshonate. The complex formed crystals of extended plate morphology under low ionic-strength conditions. X-ray diffraction data were collected to 2.8 A resolution at the ESRF. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 68.7, b = 197.7, c = 86.1 A, beta = 113.4 degrees. Preliminary structural analysis revealed the presence of one molecule in the asymmetric unit.     

Synthesis of selenium-derivatized nucleosides and oligonucleotides for X-ray crystallography

We report here the synthesis of nucleoside and oligonucleotide analogs containing selenium, which serves as an anomalous scattering center to enable MAD phase determination in nucleotide X-ray crystallography. We have developed a phase transfer approach to introduce the selenium functionality in A, C, G, T, and U nucleosides at 5'-positions. In the incorporation of the selenium functionality, the leaving groups (bromide, mesyl, and tosyl) were readily displaced by sodium selenide, sodium diselenide, and sodium methyl selenide with yields higher than 90%. Selenium-derivatized oligonucleotides have been synthesized via phosphoramidite chemistry.     

New approaches to high-throughput phasing

Recent progress in macromolecular phasing, in part stimulated by the high-throughput structural biology initiatives, has made this crucial stage of the elucidation of crystal structures easier and more automatic. A quick soak in various salts leads to the rapid incorporation of the anomalously scattering ions, suitable for phasing by MAD (multiwavelength anomalous dispersion), SAD (single-wavelength anomalous dispersion) or MIR (multiple isomorphous replacement) methods. The availability of stable synchrotron beam lines equipped with elaborate hardware control and sophisticated data processing programs makes it possible to collect very accurate diffraction data and to solve structures from the very weak anomalous signal of such atoms as sulfur or phosphorus, inherently present in macromolecules. The current progress in phasing, coupled with the parallel advances in protein crystallization, diffraction data collection and so on, suggests that, in the near future, the process of macromolecular crystal structure elucidation may become fully automatic.