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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.

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Diffraction pattern of Sso6206 collected at the ESRF.
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fig3: Diffraction pattern of Sso6206 collected at the ESRF.

Mentions: A 2.7 Å data set was collected in-house at 130 K from a single crystal mounted on a loop. The crystal was subject to a soaking in 15%(v/v) ethylene glycol in stabilization buffer before transferring to cryogenic conditions for data collection. Data was collected as 20 min 0.5° oscillations using a Rigaku HTC image-plate detector, a rotating-anode X-ray source and Osmic mirrors. The crystal-to-detector distance was 220 mm. All data were integrated using MOSFLM (Leslie, 1992 ▶) and merged with SCALA (Evans, 1997 ▶) as implemented in CCP4 (Collaborative Computational Project, Number 4, 1994 ▶). The initial indexing showed the crystals to be primitive hexagonal, with unit-cell parameters a = b = 158.1, c = 308.3 Å, α = β = 90, γ = 120°; a second native data set was collected at Daresbury which showed similar space group and resolution. A third data set was collected at the European Scientific Research Facility (ESRF) Grenoble with the same space group and unit-cell parameters and improved resolution (Fig. 3 ▶). Data-collection statistics are given in Table 1 ▶. Merging of the data indicates that the space group belongs to the higher symmetry 6/mmm Laue class. Analysis of over 100 00l axial reflections shows a clear l = 6n condition, indicating that the space group is P6122 or its enantiomorph P6522.


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)

Diffraction pattern of Sso6206 collected at the ESRF.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Diffraction pattern of Sso6206 collected at the ESRF.
Mentions: A 2.7 Å data set was collected in-house at 130 K from a single crystal mounted on a loop. The crystal was subject to a soaking in 15%(v/v) ethylene glycol in stabilization buffer before transferring to cryogenic conditions for data collection. Data was collected as 20 min 0.5° oscillations using a Rigaku HTC image-plate detector, a rotating-anode X-ray source and Osmic mirrors. The crystal-to-detector distance was 220 mm. All data were integrated using MOSFLM (Leslie, 1992 ▶) and merged with SCALA (Evans, 1997 ▶) as implemented in CCP4 (Collaborative Computational Project, Number 4, 1994 ▶). The initial indexing showed the crystals to be primitive hexagonal, with unit-cell parameters a = b = 158.1, c = 308.3 Å, α = β = 90, γ = 120°; a second native data set was collected at Daresbury which showed similar space group and resolution. A third data set was collected at the European Scientific Research Facility (ESRF) Grenoble with the same space group and unit-cell parameters and improved resolution (Fig. 3 ▶). Data-collection statistics are given in Table 1 ▶. Merging of the data indicates that the space group belongs to the higher symmetry 6/mmm Laue class. Analysis of over 100 00l axial reflections shows a clear l = 6n condition, indicating that the space group is P6122 or its enantiomorph P6522.

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