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2.7.1.30: glycerol kinase

This is an abbreviated version!
For detailed information about glycerol kinase, go to the full flat file.

Word Map on EC 2.7.1.30

Reaction

ATP
+
glycerol
=
ADP
 +
sn-glycerol 3-phosphate

Synonyms

AFUB_068560, ASTP , ATP-stimulated glucocorticoid-receptor translocation promoter , ATP: glycerol 3-phosphotransferase, ATP: glycerol-3-phosphotransferase, ATP:glycerol 3-phosphotransferase, ATP:glycerol 3-phosphotransferase , ATP:glycerol-3-phosphotransferase, GK, GK1, GK2, GK3, GK4, GK5, glcA, GLPK, glyceric kinase, glycerokinase, glycerol kinase, glycerol kinase 2, glycerol kinase 5, GLYK, GUT1, GYK, kinase, glycerol (phosphorylating), KpGlpK, More, slr1672, TbgGK, Tk-GK

ECTree

     2 Transferases
         2.7 Transferring phosphorus-containing groups
             2.7.1 Phosphotransferases with an alcohol group as acceptor
                2.7.1.30 glycerol kinase

Crystallization

Crystallization on EC 2.7.1.30 - glycerol kinase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystals are obtained by the hanging-drop technique from a solution containing 29% polyethylene glycol 400, 0.1 M sodium acetate pH 4.5, 0.1 M calcium acetate and 10% glycerol. The crystals can grow in the presence of 33% PEG 400, which allows to mount the crystals and directyl flash-cool them. Repeated flash-annealing causes a significant decrease in the averaged mosaicity along with an increase in the overall peak counts of reflections and an enhanced signal-to-noise ratio. Individual reflection-profile analysis reveales a mostly dual domain structure, showing the minimization of one domain as a result of flash-annealing.
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crystals of native and mutant enzyme with bound glycerol, hanging drop vapor diffusion method
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in complex with glycerol, ADP and the allosteric effector enzyme IIAGlc
-
in complex with glycerol, in presence and absence of fructose 1,6-diphosphate, mechanism
of wild type and mutant A65T, both in complex with glycerol and ADP, and of mutant I474D, in complex with IIAGlc
-
the crystal structure of glycerol kinase mutant G230D is determined to 2.0 A resolution using a microfluidics based crystallization platform
using modified microfluidic scale-up diffraction device, crystals form after one week at ambient temperature unter crystallization conditions 0.3 M magnesium chloride, 0.1 M TrisHCl (pH 8.5), and 20% PEG 1500. Using vapor diffusion crystallization, crystals appear after one week at ambient temperature using the crystallization conditions 0.1 M magnesium chloride, 0.1 M TrisHCl (pH 8.5), and 10% PEG 1500. Glycerol kinase of the mutant G230D crystallied in space group P21 with two tetramers of 222 point symmetry in the asu. The average B factor for the overall structure of the G230D mutant is 21.2 A2.
crystals are grown at 20°C by the sitting-drop vapour diffusion method. Native X-ray diffraction data are collected to 2.4 A resolution using synchrotron radiation at station BL44XU of SPring-8. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 217.48, c = 66.48 A. The protein is also cocrystallized with substrates and diffraction data are collected to 2.7 A resolution
purified recombinant wild-type enzyme in complex with glycerol and AMPPCP, and enzyme mutant K271E free or in complex with glycerol, sitting drop vapor diffusion method, 4-5 mg/ml protein in 5 mM Tris-HCl, pH 7.5, with or without 5 mM glycerol, and 5 mM AMPPCP, is mixed with 0.1 M HEPES, pH 8.0, containing 15% w/v PEG 1000 and 200 mM calcium acetate, 20°C, X-ray diffraction structure determination and analysis at 2.3-3.1 A resolution, molecular replacement using the structure of Tk-GK (PDB ID 2ZF5) as template, modelling
using sitting-drop vapour-diffusion method at 293 K. Native Tk-glycerol kinase crystals appear after a few days using Wizard I solution No. 25 (0.1 M Tris pH 8.5, 0.2 M MgCl2, 30% PEG 400). Diffraction spots sufficient for structural determination at high resolution are not obtained when a crystal of Tk-glycerol kinase is mounted on a CryoLoop without cryoprotectant, diffraction patterns of the crystal are improved by using Paratone-N as cryoprotectant. Native X-ray diffraction data are collected to 2.4 A resolution using synchrotron radiation at station BL44XU of SPring-8. The crystals belong to the rhombohedral space group R3, with unit-cell parameters a = b = 217.48, c = 66.48 A. Assuming the presence of two molecules in the asymmetric unit, the VM value is 2.7 A3Da-1 and the solvent content is 54.1%.
-
in complex with substrates, sitting drop vapor diffusion method, using M HEPES pH 7.5, 11% (v/v) hexane-1,6-diol, 25% (w/v) PEG400
purified recombinant His6-tagged enzyme in complex with pNPP, sitting drop method, mixing of 0.001 ml of 5 mg/mL TbgGK in 10 mM MgSO4, 20 mM NaCl, 0.001% glycerol and 10 mM MOPS, pH 6.8, with 0.001 ml of 11% 1,6-hexanediol, 25% PEG 400, and 0.1 M HEPES, pH 7.5, at 20°C, crystals of the TbgGK-pNPP complex are obtained by soaking TbgGK crystals in a cryoprotectant solution containing 5.5% 1,6-hexanediol, 40% PEG 400, and 0.1 M HEPES, pH 7.5, that is supplemented with 20 mM pNPP, at 20°C, for 6-14 h, X-ray diffraction structure determination and analysis, molecular replacement method using the protein structure of the TbgGK-ADP complex (PDB ID 3WXL) as a search model
sitting drop vapor diffusion method, using 30% (w/v) PEG 400 and 100 mM HEPES pH 7.0, at 20°C
purified recombinant His6-tagged enzyme in complex with pNPP, sitting drop method, mixing of 0.001 ml of 5 mg/mL TbgGK in 10 mM MgSO4, 20 mM NaCl, 0.001% glycerol and 10 mM MOPS, pH 6.8, with 0.001 ml of 11% 1,6-hexanediol, 25% PEG 400, and 0.1 M HEPES, pH 7.5, at 20°C, crystals of the TbgGK-pNPP complex are obtained by soaking TbgGK crystals in a cryoprotectant solution containing 5.5% 1,6-hexanediol, 40% PEG 400, and 0.1 M HEPES, pH 7.5, that is supplemented with 20 mM pNPP, at 20°C, for 6-14 h, X-ray diffraction structure determination and analysis, molecular replacement method using the protein structure of the TbgGK-ADP complex (PDB ID 3WXL) as a search model