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Purification, crystallization and preliminary X-ray diffraction analysis of a soluble variant of the monoglyceride lipase Yju3p from the yeast Saccharomyces cerevisiae.

Rengachari S, Aschauer P, Sturm C, Oberer M - Acta Crystallogr F Struct Biol Commun (2015)

Bottom Line: The protein Yju3p is the orthologue of monoglyceride lipases in the yeast Saccharomyces cerevisiae.The crystal form was orthorhombic (space group P212121), with unit-cell parameters a = 77.2, b = 108.6, c = 167.7 Å.The asymmetric unit contained four molecules with a solvent content of 46.4%.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Biology, University of Graz, Humboldtstrasse 50/3, 8010 Graz, Austria.

ABSTRACT
The protein Yju3p is the orthologue of monoglyceride lipases in the yeast Saccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space group P212121), with unit-cell parameters a = 77.2, b = 108.6, c = 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%.

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Crystals of s-Yju3p obtained by the hanging-drop method at 293 K in 0.1 M bicine/Trizma base pH 8.7, 10%(w/v) PEG 20 000, 20%(v/v) PEG MME 550, 0.03 M sodium nitrate, 0.03 M disodium hydrogen phosphate, 0.03 M ammonium sulfate. The scale bar is 200 µm in length.
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fig2: Crystals of s-Yju3p obtained by the hanging-drop method at 293 K in 0.1 M bicine/Trizma base pH 8.7, 10%(w/v) PEG 20 000, 20%(v/v) PEG MME 550, 0.03 M sodium nitrate, 0.03 M disodium hydrogen phosphate, 0.03 M ammonium sulfate. The scale bar is 200 µm in length.

Mentions: s-Yju3p crystallized as a cluster of platelets and crystals were detected after 7 days (Fig. 2 ▶). Single crystals from the cluster were harvested and flash-cooled in liquid nitrogen without the addition of further cryoprotectants (the addition of glycerol as a cryoprotectant led to the disintegration of the crystals). 270 diffraction images (oscillation angle 1°) were collected at 100 K from the best crystal diffracting to 2.4 Å resolution (Fig. 3 ▶), but the data set was cut at 2.6 Å during scaling based on the CC1/2 and Rmerge values. The data set was integrated in the primitive orthorhombic space group P212121 as verified by POINTLESS (Evans, 2006 ▶). The unit-cell parameters were a = 77.2, b = 108.6, c = 167.7 Å. According to the Matthews coefficient, the asymmetric unit contained four molecules with 46.4% solvent content [2.29 Å3 Da−1, P(tot) = 0.70; Matthews, 1968 ▶]. Phenix.xtriage revealed that the crystal form contained neither twinning nor pseudo-translational symmetry (Adams et al., 2010 ▶). Attempts to derive the phases of s-Yju3p by molecular replacement failed owing to a lack of high-identity structures. According to a BLAST search, the protein of known structure that has the highest sequence identity is MGL from H. sapiens, with a sequence identity of 24% (based on 97% query coverage). Hence, approaches leading to experimental phase determination are being pursued, i.e. the use of selenomethionine-incorporated protein for SAD and the use of heavy-atom soaking for MIR/MAD.


Purification, crystallization and preliminary X-ray diffraction analysis of a soluble variant of the monoglyceride lipase Yju3p from the yeast Saccharomyces cerevisiae.

Rengachari S, Aschauer P, Sturm C, Oberer M - Acta Crystallogr F Struct Biol Commun (2015)

Crystals of s-Yju3p obtained by the hanging-drop method at 293 K in 0.1 M bicine/Trizma base pH 8.7, 10%(w/v) PEG 20 000, 20%(v/v) PEG MME 550, 0.03 M sodium nitrate, 0.03 M disodium hydrogen phosphate, 0.03 M ammonium sulfate. The scale bar is 200 µm in length.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321484&req=5

fig2: Crystals of s-Yju3p obtained by the hanging-drop method at 293 K in 0.1 M bicine/Trizma base pH 8.7, 10%(w/v) PEG 20 000, 20%(v/v) PEG MME 550, 0.03 M sodium nitrate, 0.03 M disodium hydrogen phosphate, 0.03 M ammonium sulfate. The scale bar is 200 µm in length.
Mentions: s-Yju3p crystallized as a cluster of platelets and crystals were detected after 7 days (Fig. 2 ▶). Single crystals from the cluster were harvested and flash-cooled in liquid nitrogen without the addition of further cryoprotectants (the addition of glycerol as a cryoprotectant led to the disintegration of the crystals). 270 diffraction images (oscillation angle 1°) were collected at 100 K from the best crystal diffracting to 2.4 Å resolution (Fig. 3 ▶), but the data set was cut at 2.6 Å during scaling based on the CC1/2 and Rmerge values. The data set was integrated in the primitive orthorhombic space group P212121 as verified by POINTLESS (Evans, 2006 ▶). The unit-cell parameters were a = 77.2, b = 108.6, c = 167.7 Å. According to the Matthews coefficient, the asymmetric unit contained four molecules with 46.4% solvent content [2.29 Å3 Da−1, P(tot) = 0.70; Matthews, 1968 ▶]. Phenix.xtriage revealed that the crystal form contained neither twinning nor pseudo-translational symmetry (Adams et al., 2010 ▶). Attempts to derive the phases of s-Yju3p by molecular replacement failed owing to a lack of high-identity structures. According to a BLAST search, the protein of known structure that has the highest sequence identity is MGL from H. sapiens, with a sequence identity of 24% (based on 97% query coverage). Hence, approaches leading to experimental phase determination are being pursued, i.e. the use of selenomethionine-incorporated protein for SAD and the use of heavy-atom soaking for MIR/MAD.

Bottom Line: The protein Yju3p is the orthologue of monoglyceride lipases in the yeast Saccharomyces cerevisiae.The crystal form was orthorhombic (space group P212121), with unit-cell parameters a = 77.2, b = 108.6, c = 167.7 Å.The asymmetric unit contained four molecules with a solvent content of 46.4%.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Biology, University of Graz, Humboldtstrasse 50/3, 8010 Graz, Austria.

ABSTRACT
The protein Yju3p is the orthologue of monoglyceride lipases in the yeast Saccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space group P212121), with unit-cell parameters a = 77.2, b = 108.6, c = 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%.

Show MeSH
Related in: MedlinePlus