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Single-wavelength Anomalous Dispersion (single-wavelength + anomalous_dispersion)
Selected AbstractsThe magic triangle goes MAD: experimental phasing with a bromine derivativeACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010Tobias Beck Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br,K edge was successfully carried out. Radiation damage to the bromine,carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out. [source] Structure of a fatty acid-binding protein from Bacillus subtilis determined by sulfur-SAD phasing using in-house chromium radiationACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Jie Nan Sulfur single-wavelength anomalous dispersion (S-SAD) and halide-soaking methods are increasingly being used for ab initio phasing. With the introduction of in-house Cr X-ray sources, these methods benefit from the enhanced anomalous scattering of S and halide atoms, respectively. Here, these methods were combined to determine the crystal structure of BsDegV, a DegV protein-family member from Bacillus subtilis. The protein was cocrystallized with bromide and low-redundancy data were collected to 2.5,Å resolution using Cr,K, radiation. 17 heavy-atom sites (ten sulfurs and seven bromides) were located using standard methods. The anomalous scattering of some of the BsDegV S atoms and Br atoms was weak, thus neither sulfurs nor bromides could be used alone for structure determination using the collected data. When all 17 heavy-atom sites were used for SAD phasing, an easily interpretable electron-density map was obtained after density modification. The model of BsDegV was built automatically and a palmitate was found tightly bound in the active site. Sequence alignment and comparisons with other known DegV structures provided further insight into the specificity of fatty-acid selection and recognition within this protein family. [source] Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehydeACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Matthew W. Vetting The pentapeptide-repeat protein EfsQnr from Enterococcus faecalis protects DNA gyrase from inhibition by fluoroquinolones. EfsQnr was cloned and purified to homogeneity, but failed to produce diffraction-quality crystals in initial crystallization screens. Treatment of EfsQnr with glutaraldehyde and the strong reducing agent borane,dimethylamine resulted in a derivatized protein which produced crystals that diffracted to 1.6,Å resolution; their structure was subsequently determined by single-wavelength anomalous dispersion. Analysis of the derivatized protein using Fourier transform ion cyclotron resonance mass spectrometry indicated a mass increase of 68,Da per free amino group. Electron-density maps about a limited number of structurally ordered lysines indicated that the modification was a cyclic pentylation of free amines, producing piperidine groups. [source] Structure of T4moC, the Rieske-type ferredoxin component of toluene 4-monooxygenaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2006Luke A. Moe The structure of the Rieske-type ferredoxin (T4moC) from toluene 4-monooxygenase was determined by X-ray crystallography in the [2Fe,2S]2+ state at a resolution of 1.48,Å using single-wavelength anomalous dispersion phasing with the [2Fe,2S] center. The structure consists of ten ,-strands arranged into the three antiparallel ,-sheet topology observed in all Rieske proteins. Trp69 of T4moC is adjacent to the [2Fe,2S] centre, which displaces a loop containing the conserved Pro81 by ,8,Å away from the [2Fe,2S] cluster compared with the Pro loop in the closest structural and functional homolog, the Rieske-type ferredoxin BphF from biphenyl dioxygenase. In addition, T4moC contains five hydrogen bonds to the [2Fe,2S] cluster compared with three hydrogen bonds in BphF. Moreover, the electrostatic surface of T4moC is distinct from that of BphF. These structural differences are identified as possible contributors to the evolutionary specialization of soluble Rieske-type ferredoxins between the diiron monooxygenases and cis -dihydrodiol-forming dioxygenases. [source] Novel approach to phasing proteins: derivatization by short cryo-soaking with halidesACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2000Zbigniew Dauter A quick (less than 1,min) soak of protein crystals in a cryo-solution containing bromide or iodide anions leads to incorporation of these anomalous scatterers into the ordered solvent region around the protein molecules. These halide anions provide a convenient way of phasing through their anomalous scattering signal: bromides using multiwavelength anomalous dispersion (MAD) and bromides and/or iodides using single-wavelength anomalous dispersion (SAD) or single isomorphous replacement with anomalous scattering (SIRAS) methods. This approach has been tested successfully on four different proteins and has been used to solve the structure of a new protein of molecular weight 30,kDa. [source] Structure of Stenotrophomonas maltophilia FeoA complexed with zinc: a unique prokaryotic SH3-domain protein that possibly acts as a bacterial ferrous iron-transport activating factorACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Yi-Che Su Iron is vital to the majority of prokaryotes, with ferrous iron believed to be the preferred form for iron uptake owing to its much better solubility. The major route for bacterial ferrous iron uptake is found to be via an Feo (ferrous iron-transport) system comprising the three proteins FeoA, FeoB and FeoC. Although the structure and function of FeoB have received much attention recently, the roles played by FeoA and FeoC have been little investigated to date. Here, the tertiary structure of FeoA from Stenotrophomonas maltophilia (Sm), a vital opportunistic pathogen in immunodepressed hosts, is reported. The crystal structure of SmFeoA has been determined to a resolution of 1.7,Å using an Se single-wavelength anomalous dispersion (Se-SAD) approach. Although SmFeoA bears low sequence identity to eukaryotic proteins, its structure is found to adopt a eukaryotic SH3-domain-like fold. It also bears weak similarity to the C-terminal SH3 domain of bacterial DtxR (diphtheria toxin regulator), with some unique characteristics. Intriguingly, SmFeoA is found to adopt a unique dimer cross-linked by two zinc ions and six anions (chloride ions). Since FeoB has been found to contain a G-protein-like domain with low GTPase activity, FeoA may interact with FeoB through the SH3,G-protein domain interaction to act as a ferrous iron-transport activating factor. [source] Rv0802c from Mycobacterium tuberculosis: the first structure of a succinyltransferase with the GNAT foldACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2008Matthew W. Vetting Gene rv0802c from Mycobacterium tuberculosis encodes a 218-amino-acid protein and is annotated as a hypothetical protein with homology to GCN5-related N -acetyltransferases. The structure of Rv0802c was determined in an unliganded form to 2.0,Å resolution utilizing single-wavelength anomalous dispersion from a samarium soak that resulted in a single bound Sm3+:citrate2 complex. The structure confirms that Rv0802c exhibits the GCN5-related N -acetyltransferase fold and revealed a tetramer composed of a dimer of dimers with approximate 222 symmetry. In addition, a bound acetate ion indicated that Rv0802c may utilize a unique acyl donor for the family. The subsequent determination of the structure of Rv0802c in complex with succinyl-CoA to 2.3,Å resolution suggests that Rv0802c is the first known GCN5-related N -acetyltransferase family member to utilize succinyl-CoA as a substrate. [source] Crystallization and preliminary crystallographic analysis of the cellulose biosynthesis-related protein CMCax from Acetobacter xylinumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2005Shin Kawano The cellulose biosynthesis-related protein CMCax from Acetobacter xylinum was overexpressed in Escherichia coli, purified and crystallized. Single crystals of selenomethionine (SeMet) substituted CMCax were obtained by the hanging-drop vapour-diffusion method at 293,K, primarily using polyethylene glycol 4000 as a precipitant. The crystals belong to the primitive hexagonal space group P61 or P65, with unit-cell parameters a = b = 89.1, c = 94.2,Å. The predicted Matthews coefficient (VM) value is 3.0,Å3,Da,1 for one CMCax monomer in the asymmetric unit. A single-wavelength anomalous dispersion (SAD) data set was collected to a resolution of 2.3,Å using synchrotron radiation. [source] | ||||||||||