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Sitting-drop Method (sitting-drop + method)
Selected AbstractsCrystallization and preliminary X-ray diffraction analysis of homing endonuclease I- Tsp061IACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2004Takahito Imagawa Two crystal forms, rhombohedral and hexagonal, of a homing endonuclease from Thermoproteus sp. IC-061 (I- Tsp0611) were obtained by the hanging-drop and sitting-drop method, respectively. The hexagonal crystals belong to space group P6322, with unit-cell parameters a = b = 111.4, c = 97.6,Å, and diffract to 3.2,Å resolution on beamline BL44 at SPring-8 (Harima, Japan). The rhombohedral crystals belong to space group R32, with unit-cell parameters a = b = 95.4, c = 192.9,Å, and diffract to 2.7,Å resolution using a Cu,K, rotating-anode generator with an R-AXIS VII detector. The crystal asymmetric unit contained one protein molecule and the solvent contents of the two crystal forms were estimated to be 68.3 and 67.6% by volume, respectively. [source] Incorporation of methyl-protonated valine and leucine residues into deuterated ocean pout type III antifreeze protein: expression, crystallization and preliminary neutron diffraction studiesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Isabelle Petit-Haertlein Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-acid precursor ,-ketoisovalerate. Here, the successful methyl protonation (methyl reverse-labelling) of leucine and valine residues in AFP is reported. Methyl-protonated AFP was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Crystals were grown in D2O buffer by the sitting-drop method. Preliminary neutron Laue diffraction at 293,K using LADI-III at ILL showed in a few 24,h exposures a very low background and clear small spots up to a resolution of 1.80,Å from a crystal of dimensions 1.60 × 0.38 × 0.38,mm corresponding to a volume of 0.23,mm3. [source] Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze proteinACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Isabelle Petit-Haertlein The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D2O by the sitting-drop method. Preliminary neutron Laue diffraction at 293,K using LADI-III at ILL showed that with a few exposures of 24,h a very low background and clear small spots up to a resolution of 1.85,Å were obtained using a `radically small' perdeuterated AFP D crystal of dimensions 0.70 × 0.55 × 0.35,mm, corresponding to a volume of 0.13,mm3. [source] A simple technique to convert sitting-drop vapor diffusion into hanging-drop vapor diffusion by solidifying the reservoir solution with agaroseJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 5 2009Tae Woong Whon A simple protocol to convert sitting-drop vapor-diffusion plating into a hanging-drop vapor-diffusion experiment in protein crystallization is reported. After making a sitting-drop plate, agarose solution was added to solidify the reservoir solution, and the plates were incubated upside down. Crystallization experiments with hen egg white lysozyme, thaumatin and glucose isomerase showed that the `upside-down sitting-drop' method could produce single crystals with all the benefits of the hanging-drop crystallization method. [source] | ||||||||||