Limits...
Overexpression, purification, crystallization and data collection of Sulfolobus solfataricus Sso6206, a novel highly conserved protein.

McEwan AR, Liu H, Oke M, Carter L, Powers H, Dorward M, McMahon SA, White MF, Naismith JH - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2006)

Bottom Line: The protein crystallizes in space group P6(1/5)22, with unit-cell parameters a = b = 157.8, c = 307.3 A.Crystals of selenomethionine-variant protein have not yet been obtained.Interestingly, crystal packing, gel filtration and mass spectrometry all suggest the native protein forms a multi-subunit oligomer consisting of >9 subunits.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Biomolecular Sciences, The University, St Andrews KY16 9ST, Scotland.

ABSTRACT
Sso6206, a 10.5 kDa protein from Sulfolobus solfataricus, has been overexpressed, purified and crystallized. The protein crystallizes in space group P6(1/5)22, with unit-cell parameters a = b = 157.8, c = 307.3 A. The crystals are hexagonal bipyramids and a data set has been collected to 2.4 A resolution. Molecular replacement cannot be attempted as no convincing model can be identified. Crystals of selenomethionine-variant protein have not yet been obtained. Interestingly, crystal packing, gel filtration and mass spectrometry all suggest the native protein forms a multi-subunit oligomer consisting of >9 subunits.

Show MeSH
Crystal of Sso6206.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2197187&req=5

fig2: Crystal of Sso6206.

Mentions: Initial conditions were obtained using a sitting-drop vapour-diffusion screen of commercial sparse-matrix crystallization conditions. Three protein concentrations (6, 3 and 1 mg ml−1) were screened at 293 K with a drop size of 0.2 µl (containing 0.1 µl protein solution and 0.1 µl precipitant solution) prepared using a nanodrop crystallization robot (Cartesian HoneyBee) as a part of the Hamilton–Thermo Rhombix system. Optimization of the initial 12 most promising hits, including at least one at each concentration, were performed to confirm that the crystals contained protein. The largest protein crystals were obtained using the hanging-drop vapour-diffusion method (2 µl + 2 µl) at 293 K. Improved crystals could be obtained from a number of conditions; however, the best crystals, as judged by size and regular shape, were obtained from variation of Wizard I (Emerald Biosystems) condition Nos. 31 [20%(w/v) PEG 8000, 0.1 M phosphate–citrate pH 4.2, 0.2 M NaCl] and 36 (1.0 M sodium citrate, 0.1 M imidazole pH 8.0). The crystals obtained from both conditions were of the protein and had hexagonal bipyramidal morphology (0.4 × 0.4 × 0.4 mm). The crystals appear as a spherical precipitate within 2 d and gradually develop more distinct edges over the course of a month. Diffraction analysis showed that the crystals obtained from a precipitant of 0.6 M sodium citrate, 0.1 mM imidazole pH 7.75 with a protein concentration of 5 mg ml−1 were optimal (Fig. 2 ▶). We have been able to express the selenomethionine variant of the protein using methioinine inhibition (Doublié, 1997 ▶). The protein was purified in the same way as the native protein. Mass spectrometry confirmed full incorporation of selenium and did not indicate that any oxidation had occurred. The selenomethionine protein appeared to be homogenous by mass spectrometry and SDS–PAGE gel electrophoresis. However, we have so far been unable to crystallize the selenomethionine variant of the protein either using the native conditions or in a re-screen of sparse-matrix conditions. Circular-dichroism spectrometry suggests the selenomethionine protein is not significantly different and the protein has a similar profile to the native protein on size-exclusion chromatography.


Overexpression, purification, crystallization and data collection of Sulfolobus solfataricus Sso6206, a novel highly conserved protein.

McEwan AR, Liu H, Oke M, Carter L, Powers H, Dorward M, McMahon SA, White MF, Naismith JH - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2006)

Crystal of Sso6206.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2197187&req=5

fig2: Crystal of Sso6206.
Mentions: Initial conditions were obtained using a sitting-drop vapour-diffusion screen of commercial sparse-matrix crystallization conditions. Three protein concentrations (6, 3 and 1 mg ml−1) were screened at 293 K with a drop size of 0.2 µl (containing 0.1 µl protein solution and 0.1 µl precipitant solution) prepared using a nanodrop crystallization robot (Cartesian HoneyBee) as a part of the Hamilton–Thermo Rhombix system. Optimization of the initial 12 most promising hits, including at least one at each concentration, were performed to confirm that the crystals contained protein. The largest protein crystals were obtained using the hanging-drop vapour-diffusion method (2 µl + 2 µl) at 293 K. Improved crystals could be obtained from a number of conditions; however, the best crystals, as judged by size and regular shape, were obtained from variation of Wizard I (Emerald Biosystems) condition Nos. 31 [20%(w/v) PEG 8000, 0.1 M phosphate–citrate pH 4.2, 0.2 M NaCl] and 36 (1.0 M sodium citrate, 0.1 M imidazole pH 8.0). The crystals obtained from both conditions were of the protein and had hexagonal bipyramidal morphology (0.4 × 0.4 × 0.4 mm). The crystals appear as a spherical precipitate within 2 d and gradually develop more distinct edges over the course of a month. Diffraction analysis showed that the crystals obtained from a precipitant of 0.6 M sodium citrate, 0.1 mM imidazole pH 7.75 with a protein concentration of 5 mg ml−1 were optimal (Fig. 2 ▶). We have been able to express the selenomethionine variant of the protein using methioinine inhibition (Doublié, 1997 ▶). The protein was purified in the same way as the native protein. Mass spectrometry confirmed full incorporation of selenium and did not indicate that any oxidation had occurred. The selenomethionine protein appeared to be homogenous by mass spectrometry and SDS–PAGE gel electrophoresis. However, we have so far been unable to crystallize the selenomethionine variant of the protein either using the native conditions or in a re-screen of sparse-matrix conditions. Circular-dichroism spectrometry suggests the selenomethionine protein is not significantly different and the protein has a similar profile to the native protein on size-exclusion chromatography.

Bottom Line: The protein crystallizes in space group P6(1/5)22, with unit-cell parameters a = b = 157.8, c = 307.3 A.Crystals of selenomethionine-variant protein have not yet been obtained.Interestingly, crystal packing, gel filtration and mass spectrometry all suggest the native protein forms a multi-subunit oligomer consisting of >9 subunits.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Biomolecular Sciences, The University, St Andrews KY16 9ST, Scotland.

ABSTRACT
Sso6206, a 10.5 kDa protein from Sulfolobus solfataricus, has been overexpressed, purified and crystallized. The protein crystallizes in space group P6(1/5)22, with unit-cell parameters a = b = 157.8, c = 307.3 A. The crystals are hexagonal bipyramids and a data set has been collected to 2.4 A resolution. Molecular replacement cannot be attempted as no convincing model can be identified. Crystals of selenomethionine-variant protein have not yet been obtained. Interestingly, crystal packing, gel filtration and mass spectrometry all suggest the native protein forms a multi-subunit oligomer consisting of >9 subunits.

Show MeSH