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Derivative Crystals (derivative + crystal)
Selected AbstractsHigh-phasing-power lanthanide derivatives: taking advantage of ytterbium and lutetium for optimized anomalous diffraction experiments using synchrotron radiationACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2003É. Girard Ytterbium and lutetium are well suited for optimized anomalous diffraction experiments using synchrotron radiation. Therefore, two lanthanide complexes Yb-HPDO3A and Lu-HPDO3A have been produced that are similar to the Gd-HPDO3A complex already known to give good derivative crystals. Derivative crystals of hen egg-white lysozyme were obtained by co-crystallization using 100,mM solutions of each lanthanide complex. De novo phasing has been carried out using single-wavelength anomalous diffraction on data sets collected on each derivative crystal at the LIII absorption edge of the corresponding lanthanide ( = 28,e,). A third data set was collected on a Lu-HPDO3A derivative crystal at the Se,K absorption edge with = 10,e,. The structures were refined and compared with the known structure of the Gd-HPDO3A lysozyme derivative. The quality of the experimental electron-density maps allows easy model building. With LIII absorption edges at shorter wavelengths than the gadolinium absorption edge, lutetium and ytterbium, when chelated by a ligand such as HPDO3A, form lanthanide complexes that are especially interesting for synchrotron-radiation experiments in structural biology. [source] Crystallization and preliminary crystallographic analysis of extracellular fragment X3 of YWK-II/APPH: a human sperm membrane protein related to the Alzheimer ,A4-amyloid precursor proteinACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2003Wangjun Hu Crystals of extracellular fragment X3 of a human sperm membrane protein YWK-II/APPH have been grown at 291,K using 8% PEG 4000 as precipitant by the vapour-diffusion method. The diffraction pattern of the crystal extends to 2.9,Å resolution at 100,K using Cu,K, radiation in-house. The crystals belong to space group P21, with unit-cell parameters a = 46.0, b = 43.7, c = 90.2,Å, , = , = 90.0, , = 106.6°. Furthermore, a selenomethionine (SeMet) derivative of the protein was overexpressed in the same expression system and was purified in a reducing environment. The derivative crystals were obtained under similar conditions. Subsequently, a single-wavelength data set was collected to 2.38,Å resolution from the derivative crystal at ESRF. The crystals belong to space group P21, with unit-cell parameters a = 46.2, b = 44.0, c = 88.3,Å, , = , = 90.0, , = 103.6°. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (VM) of 2.8,Å3,Da,1 and a solvent content of 56.4% by volume. [source] Atomic resolution structure of Escherichia coli dUTPase determined ab initioACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2001A. González Cryocooled crystals of a mercury complex of Escherichia coli dUTPase diffract to atomic resolution. Data to 1.05,Å resolution were collected from a derivative crystal and the structure model was derived from a Fourier map with phases calculated from the coordinates of the Hg atom (one site per subunit of the trimeric enzyme) using the program ARP/wARP. After refinement with anisotropic temperature factors a highly accurate model of the bacterial dUTPase was obtained. Data to 1.45,Å from a native crystal were also collected and the 100,K structures were compared. Inspection of the refined models reveals that a large part of the dUTPase remains rather mobile upon freezing, with 14% of the main chain being totally disordered and with numerous side chains containing disordered atoms in multiple discrete conformations. A large number of those residues surround the active-site cavity. Two glycerol molecules (the cryosolvent) occupy the deoxyribose-binding site. Comparison between the native enzyme and the mercury complex shows that the active site is not adversely affected by the binding of mercury. An unexpected effect seems to be a stabilization of the crystal lattice by means of long-range interactions, making derivatization a potentially useful tool for further studies of inhibitor,substrate-analogue complexes of this protein at very high resolution. [source] Autolabo: an automated system for ligand-soaking experiments with protein crystalsJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 4 2010Michihiro Sugahara Ligand soaking of protein crystals is important for the preparation of heavy-atom derivative crystals for experimental phasing as well as for large-scale ligand screening in pharmaceutical developments. To facilitate laborious large-scale ligand screening, to reduce the risk of human contact with hazardous ligand reagents and to increase the success rate of the soaking experiments, a protein crystallization robot `Autolabo' has been developed and implemented in the high-throughput crystallization-to-structure pipeline at RIKEN SPring-8 Center. The main functions of this robotic system are the production of protein crystals for experiments, the ligand soaking of these crystals and the observation of soaked crystals. The separate eight-channel dispensers of Autolabo eliminate the cross-contamination of reagents which should be strictly avoided in the ligand-soaking experiment. Furthermore, the automated approach reduces physical damage to crystals during experiments when compared with the conventional manual approach, and thereby has the potential to yield better quality diffraction data. Autolabo's performance as a ligand-soaking system was evaluated with a crystallization experiment on ten proteins from different sources and a heavy-atom derivatization experiment on three proteins using a versatile cryoprotectant containing heavy-atom reagents as ligands. The crystallization test confirmed reliable crystal reproduction in a single condition and the capability for crystallization with nucleants to improve crystal quality. Finally, Autolabo reproducibly derivatized the test protein crystals with sufficient diffraction quality for experimental phasing and model building, indicating a high potentiality of this automated approach in ligand-soaking experiments. [source] A dipicolinate lanthanide complex for solving protein structures using anomalous diffractionACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010Guillaume Pompidor Tris-dipicolinate lanthanide complexes were used to prepare derivative crystals of six proteins: hen egg-white lysozyme, turkey egg-white lysozyme, thaumatin from Thaumatococcus daniellii, urate oxidase from Aspergillus flavus, porcine pancreatic elastase and xylanase from Trichoderma reesei. Diffraction data were collected using either synchrotron radiation or X-rays from a laboratory source. In all cases, the complex turned out to be bound to the protein and the phases determined using the anomalous scattering of the lanthanide led to high-quality electron-density maps. The binding mode of the complex was characterized from the refined structures. The lanthanide tris-dipicolinate was found to bind through interactions between carboxylate groups of the dipicolinate ligands and hydrogen-bond donor groups of the protein. In each binding site, one enantiomeric form of the complex is selected from the racemic solution according to the specific site topology. For hen egg-white lysozyme and xylanase, derivative crystals obtained by cocrystallization belonged to a new monoclinic C2 crystal form that diffracted to high resolution. [source] Heavy-atom derivatives in lipidic cubic phases: results on hen egg-white lysozyme tetragonal derivative crystals with Gd-HPDO3A complexACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2004Éric Girard Gd-HPDO3A, a neutral gadolinium complex, is a good candidate for obtaining heavy-atom-derivative crystals by the lipidic cubic phase crystallization method known to be effective for membrane proteins. Gadolinium-derivative crystals of hen egg-white lysozyme were obtained by co-crystallizing the protein with 100,mM Gd-HPDO3A in a monoolein cubic phase. Diffraction data were collected to a resolution of 1.7 Å using Cu,K, radiation from a rotating-anode generator. Two binding sites of the gadolinium complex were located from the strong gadolinium anomalous signal. The Gd-atom positions and their refined occupancies were found to be identical to those found in derivative crystals of hen egg-white lysozyme obtained by co-crystallizing the protein with 100,mM Gd-HPDO3A using the hanging-drop technique. Moreover, the refined structures are isomorphous. The lipidic cubic phase is not disturbed by the high concentration of Gd-HPDO3A. This experiment demonstrates that a gadolinium complex, Gd-HPDO3A, can be used to obtain derivative crystals by the lipidic cubic phase crystallization method. Further studies with membrane proteins that are known to crystallize in lipidic cubic phases will be undertaken with Gd-HPDO3A and other Gd complexes to test whether derivative crystals with high Gd-site occupancies can be obtained. [source] High-phasing-power lanthanide derivatives: taking advantage of ytterbium and lutetium for optimized anomalous diffraction experiments using synchrotron radiationACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2003É. Girard Ytterbium and lutetium are well suited for optimized anomalous diffraction experiments using synchrotron radiation. Therefore, two lanthanide complexes Yb-HPDO3A and Lu-HPDO3A have been produced that are similar to the Gd-HPDO3A complex already known to give good derivative crystals. Derivative crystals of hen egg-white lysozyme were obtained by co-crystallization using 100,mM solutions of each lanthanide complex. De novo phasing has been carried out using single-wavelength anomalous diffraction on data sets collected on each derivative crystal at the LIII absorption edge of the corresponding lanthanide ( = 28,e,). A third data set was collected on a Lu-HPDO3A derivative crystal at the Se,K absorption edge with = 10,e,. The structures were refined and compared with the known structure of the Gd-HPDO3A lysozyme derivative. The quality of the experimental electron-density maps allows easy model building. With LIII absorption edges at shorter wavelengths than the gadolinium absorption edge, lutetium and ytterbium, when chelated by a ligand such as HPDO3A, form lanthanide complexes that are especially interesting for synchrotron-radiation experiments in structural biology. [source] Gd-HPDO3A, a complex to obtain high-phasing-power heavy-atom derivatives for SAD and MAD experiments: results with tetragonal hen egg-white lysozymeACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2002Éric Girard A neutral gadolinium complex, Gd-HPDO3A, is shown to be a good candidate to use to obtain heavy-atom derivatives and solve macromolecular structures using anomalous dispersion. Tetragonal crystals of a gadolinium derivative of hen egg-white lysozyme were obtained by co-crystallization using different concentrations of the complex. Diffraction data from three derivative crystals (100, 50 and 10,mM) were collected to a resolution of 1.7,Å using Cu,K, radiation from a rotating anode. Two strong binding sites of the gadolinium complex to the protein were located from the gadolinium anomalous signal in both the 100 and 50,mM derivatives. A single site is occupied in the 10,mM derivative. Phasing using the anomalous signal at a single wavelength (SAD method) leads to an electron-density map of high quality. The structure of the 100,mM derivative has been refined. Two molecules of the gadolinium complex are close together. Both molecules are located close to tryptophan residues. Four chloride ions were found. The exceptional quality of the SAD electron-density map, only enhanced by solvent flattening, suggests that single-wavelength anomalous scattering with the Gd-HPDO3A complex may be sufficient to solve protein structures of high molecular weight by synchrotron-radiation experiments, if not by laboratory experiments. [source] Crystallization and heavy-atom derivatization of StHsp14.0, a small heat-shock protein from Sulfolobus tokodaiiACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Takuro Hayashi Small heat-shock proteins (sHsps) bind and stabilize proteins denatured by heat or other stresses in order to prevent unfavourable protein aggregation. StHsp14.0 is an sHsp found in the acidothermophilic archaeon Sulfolobus tokodaii. A variant of StHsp14.0 was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted X-rays to 1.85,Å resolution and belonged to space group P21212, with unit-cell parameters a = 40.4, b = 61.1, c = 96.1,Å. The VM value was estimated to be 2.1,Å3,Da,1, assuming the presence of two molecules in the asymmetric unit. Heavy-atom derivative crystals were prepared successfully by the cocrystallization method and are isomorphic to native crystals. [source] Purification, crystallization and preliminary X-ray diffraction analysis of a plant subtilaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Rolf Rose The subtilase SBT3 from Solanum lycopersicum (tomato) was purified from a tomato cell culture and crystallized using the sitting-drop vapour-diffusion method. A native data set was collected to 2.5,Å resolution at 100,K using synchrotron radiation. For experimental phasing, CsCl-derivative and tetrakis(acetoxymercuri)methane (TAMM) derivative crystals were employed for MIRAS phasing. Three caesium sites and one TAMM site were identified, which allowed solution of the structure. [source] Crystallization and preliminary X-ray analysis of a novel Kunitz-type kallikrein inhibitor from Bauhinia bauhinioidesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2005Marcos Vicente de A. S. Navarro A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293,K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87,Å resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P212121, with unit-cell parameters a = 46.70, b = 64.14, c = 59.24,Å. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (VM = 2.5,Å3,Da,1). Iodinated crystals were prepared and a derivative data set was also collected at 2.1,Å resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5,M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method. [source] | ||||||||||