Cloning, expression, purification, crystallization and preliminary X-ray diffraction data of the Pyrococcus horikoshii RadA intein - PubMed (original) (raw)

Cloning, expression, purification, crystallization and preliminary X-ray diffraction data of the Pyrococcus horikoshii RadA intein

Andrzej Lyskowski et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011.

Abstract

The RadA intein from the hyperthermophilic archaebacterium Pyrococcus horikoshii was cloned, expressed and purified for subsequent structure determination. The protein crystallized rapidly in several conditions. The best crystals, which diffracted to 1.75 Å resolution, were harvested from drops consisting of 0.1 M HEPES pH 7.5, 3.0 M NaCl and were cryoprotected with Paratone-N before flash-cooling. The collected data were processed in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 58.1, b = 67.4, c = 82.9 Å. Molecular replacement with Rosetta using energy- and density-guided structure optimization provided the initial solution, which is currently under refinement.

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Figures

Figure 1

The insertion site of the _Pho_RadA intein within the _Pho_RadA protein. The primary structure of the _Pho_RadA intein is also shown. The Walker A motif is highlighted in grey.

Figure 2

Diffraction image of a _Pho_RadA intein crystal. The image was prepared using the ADXV program (

http://www.scripps.edu/\~arvai/adxv.html

). The resolution at the edge of the image is 1.75 Å. The data were collected on the ESRF ID23-1 beamline using an ADSC Quantum Q315R detector.

Figure 3

Crystals of the _Pho_RadA intein. (a, b) Crystals from the initial screen grown using (a) 0.1 M HEPES pH 7.5 and 3 M NaCl and (b) 0.1 M HEPES pH 7.0, 15% Tacsimate pH 7.0 and 2% PEG 3350. (c, d) Crystals grown from the grid optimization conditions. (c) 0.1 M HEPES pH 7.0, 15.2% Tacsimate pH 7.0. (d) 0.1 M HEPES pH 7.0, 10.4% Tacsimate pH 7.0 and 3.0% PEG 3350.

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Cloning, expression, purification, crystallization and preliminary X-ray diffraction data of the Pyrococcus horikoshii RadA intein - PubMed (original) (raw)