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Iverson Lab

Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB.

AUTHORS

Pyburn TMTasia M , Yankovskaya V Victoria , Bensing BA Barbara A , Cecchini G Gary , Sullam PM Paul M , Iverson TM T M . Acta crystallographica. Section F, Structural biology and crystallization communications. 2010 11 1; 66(Pt 11). 1503-7

ABSTRACT

The carbohydrate-binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspB(BR)) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspB(BR) buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspB(BR) in each buffer. While both sets of conditions supported crystal growth in space group P2(1)2(1)2(1), the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 Å for crystal form 1 and a = 34.6, b = 98.3, c = 99.0 Å for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall R(merge) value of 0.04 and an R(merge) value of 0.33 in the highest resolution shell.

The carbohydrate-binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspB(BR)) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspB(BR) buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspB(BR) in each buffer. While both sets of conditions supported crystal growth in space group P2(1)2(1)2(1), the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 Å for crystal form 1 and a = 34.6, b = 98.3, c = 99.0 Å for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall R(merge) value of 0.04 and an R(merge) value of 0.33 in the highest resolution shell.