Multiwavelength Anomalous Dispersion Phasing Research Articles

X-ray-induced debromination of nucleic acids at the Br K absorption edge and implications for MAD phasing.

Multi-wavelength anomalous dispersion (MAD) using brominated derivatives is considered a common and convenient technique for solving chemically synthesized nucleic acid structures. Here, it is shown that a relatively moderate X-ray dose (of the order of 5 x 10(15) photons mm(-2)) can induce sufficient debromination to prevent structure determination. The decrease in bromine occupancy with radiation dose can be accounted for by a simple exponential, with an estimated rate constant at the absorption-peak wavelength, 7.4 (0.8) MGy, that is not significantly different from its value at the absorption-edge wavelength, 9.2 (2.6) MGy (the given e.s.d.s assess the relative closeness of the two values, not their absolute accuracy, which is probably worse). Chemically, these results (and others) are consistent with bromine cleavage resulting from direct photodissociation and/or from the action of free electrons, rather than from the action of hydroxyl radicals originating from water dissociation. The free bromine species (Br(-)) diffuse too quickly, even in amorphous ice around 100 K, to allow the determination of a diffusion coefficient. From a practical point of view, it is suggested that a single data collection with a crystal consisting of iodinated instead of brominated derivatives could provide both anomalous scattering and SIR phase information by the progressive cleavage of iodine.

Insights into Ligand Binding and Catalysis of a Central Step in NAD+ Synthesis: STRUCTURES OF METHANOBACTERIUM THERMOAUTOTROPHICUMNMN ADENYLYLTRANSFERASE COMPLEXES

Nicotinamide mononucleotide adenylyltransferase (NMNATase) catalyzes the linking of NMN(+) or NaMN(+) with ATP, which in all organisms is one of the common step in the synthesis of the ubiquitous coenzyme NAD(+), via both de novo and salvage biosynthetic pathways. The structure of Methanobacterium thermoautotrophicum NMNATase determined using multiwavelength anomalous dispersion phasing revealed a nucleotide-binding fold common to nucleotidyltransferase proteins. An NAD(+) molecule and a sulfate ion were bound in the active site allowing the identification of residues involved in product binding. In addition, the role of the conserved (16)HXGH(19) active site motif in catalysis was probed by mutagenic, enzymatic and crystallographic techniques, including the characterization of an NMN(+)/SO4(2-) complex of mutant H19A NMNATase.

An efficient method for solving RNA structures: MAD phasing by replacing magnesium with zinc.

The structure of a 46-nucleotide RNA complex has been successfully solved using multi-wavelength anomalous dispersion (MAD) at the zinc K edge. Taking advantage of the eight magnesium-binding sites, it has been shown that for five of them magnesium could be replaced by zinc. This resulted in an excellent 2.0 A MAD electron-density map. Zinc, in common with some other transition metals, is able to replace magnesium in RNA structures, but zinc has the advantage of its K edge being ideally located at 1.284 A. As most RNA molecules contain magnesium-binding sites, it is suggested that this method could be a valuable alternative to the use of bromo derivatives of bases, which is limited to chemically synthesizable and thus rather short RNA sequences.

Combination of methods used in the structure solution of pyruvate:ferredoxin oxidoreductase from two crystal forms.

The structure of the homodimeric 267 kDa pyruvate:ferredoxin oxidoreductase (PFOR) of Desulfovibrio africanus was solved with data from two crystals forms, both containing two monomers per asymmetric unit. Phases were obtained from multiwavelength anomalous dispersion (MAD), solvent flattening (SF), molecular replacement (MR) using a 5 A resolution electron-density search model, multiple isomorphous replacement (MIR) and, finally, electron-density averaging (DA) procedures. It is shown how the combination of all these techniques was used to overcome problems arising from incompleteness of MAD data and weak phasing power of MIR data. A real-space refinement (RSR) procedure is described to improve MR solutions and obtain very accurate protein envelopes and non-crystallographic symmetry (NCS) transformations from 5 A resolution phase information. These were crucial for the phase extension to high resolution by DA methods.

Two-wavelength MAD phasing: in search of the optimal choice of wavelengths.

The multiwavelength anomalous dispersion (MAD) method is increasingly being used to determine protein crystal structures. In theory, data collection at two wavelengths is sufficient for the determination of MAD phases, but three or even more wavelengths are used most often. In this paper, the results of the phasing procedure using only two wavelengths for proteins containing different types of anomalous scatterers are analyzed. In these cases, it is shown that this approach leads to interpretable maps, similar in quality to those obtained with data collected at three wavelengths, provided that the wavelengths are chosen so as to give a large contrast in the real part of the anomalous scattering factor f. The consequences for a rational MAD data-collection strategy are discussed.

The molecular structure of green fluorescent protein.

The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods. The protein is in the shape of a cylinder, comprising 11 strands of beta-sheet with an alpha-helix inside and short helical segments on the ends of the cylinder. This motif, with beta-structure on the outside and alpha-helix on the inside, represents a new protein fold, which we have named the beta-can. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr66 with reduction of its C alpha-C beta bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests specific side chains that could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.

Crystal structure of the rat liver fructose-2,6-bisphosphatase based on selenomethionine multiwavelength anomalous dispersion phases.

The crystal structure of the recombinant fructose-2,6-bisphosphatase domain, which covers the residues between 251 and 440 of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was determined by multiwavelength anomalous dispersion phasing and refined at 2.5 A resolution. The selenomethionine-substituted protein was induced in the methionine auxotroph, Escherichia coli DL41DE3, purified, and crystallized in a manner similar to that of the native protein. Phase information was calculated using the multiwavelength anomalous dispersion data collected at the X-ray wavelengths near the absorption edge of the K-shell alpha electrons of selenium. The fructose-2,6-bisphosphatase domain has a core alpha/beta structure which consists of six stacked beta-strands, four parallel and two antiparallel. The core beta-sheet is surrounded by nine alpha-helices. The catalytic site, as defined by a bound phosphate ion, is positioned near the C-terminal end of the beta-sheet and close to the N-terminal end of an alpha-helix. The active site pocket is funnel-shaped. The narrow opening of the funnel is wide enough for a water molecule to pass. The key catalytic residues, including His7, His141, and Glu76, are near each other at the active site and probably function as general acids and/or bases during a catalytic cycle. The inorganic phosphate molecule is bound to an anion trap formed by Arg6, His7, Arg56, and His141. The core structure of the Fru-2,6-P2ase is similar to that of the yeast phosphoglycerate mutase and the rat prostatic acid phosphatase. However, the structure of one of the loops near the active site is completely different from the other family members, perhaps reflecting functional differences and the nanomolar range affinity of Fru-2,6-P2ase for its substrate. The imidazole rings of the two key catalytic residues, His7 and His141, are not parallel as in the yeast phosphoglycerate mutase. The crystal structure is used to interpret the existing chemical data already available for the bisphosphatase domain. In addition, the crystal structure is compared with two other proteins that belong to the histidine phosphatase family.

MAD Phasing Strategies Explored with a Brominated Oligonucleotide Crystal at 1.65Å Resolution

The crystal structure of a brominated oligonucleotide d(CGCG(Br)CG), chemical formula C(114)N(48)O(68)P(10)Br(2), has been analysed by multiwavelength anomalous dispersion (MAD) methods. The oligonucleotide crystallizes in space group P2(1)2(1)2(1) with a = 17.97, b = 30.98, c = 44.85 A, alpha = beta = gamma 90 degrees . Data to a resolution of 1.65 A were collected at four wavelengths about the K-absorption edge of the bromine atom (lambda(1) = 0.9323 A, a reference wavelength at the long-wavelength side of the edge; lambda(2) = 0.9192 A, at the absorption-edge inflection point; lambda(3) = 0.9185 A, at the ;white line' absorption maximum; lambda(4) = 0.8983 A, a reference wavelength at the short-wavelength side) using synchrotron radiation at Station PX9.5, SRS, Daresbury. Multiwavelength data could be collected on a single-crystal as the sample was radiation stable. Anomalous and dispersive Patterson maps were readily interpretable to give the bromine anomalous scatterer positions. Phase calculations to 1.65 A, resolution, using all four wavelengths, gave a figure of merit of 0.825 for 2454 reflections. The electron-density map was readily interpretable showing excellent connectivity for the sugar/phosphate backbone and each base was easily characterized. The two nucleotide strands paired up as expected in an antiparallel Watson-Crick-type manner. The structure was refined to 1.65 A using all the data (R-factor = 17.0% based on 3151 reflections, with a data-to-parameter ratio of 2.6). In addition to the four-wavelength analysis, a variety of other phasing strategies, and the associated quality of the resulting electron-density maps, were compared. These included use of either of the reference wavelength data sets in the two possible three-wavelength phasing combinations to assess their relative effectiveness. Moreover, the time dependence upon measuring the Bijvoet differences and its effect upon phasing was also investigated. Finally, the use of only two wavelengths, including Friedel pairs, is demonstrated (the theoretical minimum case); this is of particular interest when considering overall beam time needs and is clearly a feasible experimental strategy, as shown here.

Structure of the Calcium-Dependent Lectin Domain from a Rat Mannose-Binding Protein Determined by MAD Phasing

Calcium-dependent (C-type) animal lectins participate in many cell surface recognition events mediated by protein-carbohydrate interactions. The C-type lectin family includes cell adhesion molecules, endocytic receptors, and extracellular matrix proteins. Mammalian mannose-binding proteins are C-type lectins that function in antibody-independent host defense against pathogens. The crystal structure of the carbohydrate-recognition domain of a rat mannose-binding protein, determined as the holmium-substituted complex by multiwavelength anomalous dispersion (MAD) phasing, reveals an unusual fold consisting of two distinct regions, one of which contains extensive nonregular secondary structure stabilized by two holmium ions. The structure explains the conservation of 32 residues in all C-type carbohydrate-recognition domains, suggesting that the fold seen here is common to these domains. The strong anomalous scattering observed at the Ho LIII edge demonstrates that traditional heavy atom complexes will be generally amenable to the MAD phasing method.

Physical characterization and crystallization of the carbohydrate-recognition domain of a mannose-binding protein from rat.

A portion of rat mannose-binding protein A (MBP-A), a Ca(2+)-dependent animal lectin, has been overproduced in a bacterial expression system, biochemically characterized, and crystallized. A fragment corresponding to the COOH-terminal 115 residues of native MBP-A, produced by subtilisin digestion of the bacterially expressed protein, contains the carbohydrate-recognition domain (CRD). Gel filtration, chemical cross-linking, and crystallographic self-rotation function analyses indicate that the subtilisin fragment is a dimer, although the complete bacterially expressed fragment, containing the neck and CRD of MBP-A, is a trimer. Crystals of the minimal CRD, obtained only as a complex with a Man6GlcNAc2Asn glycopeptide, diffract to Bragg spacings of at least 1.7 A. Several trivalent lanthanide ions (Ln3+) can substitute for Ca2+, as assessed by their ability to support carbohydrate binding and to protect the CRD from proteolysis in a manner similar to that observed for Ca2+. These assays indicate that Ln2+ binds about 30 times more tightly than Ca2+ to the CRD, and that two Ca2+ or Ln3+ bind to each monomer, a result confirmed by determination of the Ho3+ positions in a Ho(3+)-containing crystal of the CRD. Crystals grown in the presence of Ln3+ belong to different space groups from those obtained with Ca2+ and are therefore not useable for traditional crystallographic phase determination methods, but are well-suited for high resolution structure determination by multiwavelength anomalous dispersion phasing.