Crystallization and preliminary diffraction data analysis of both single and pseudo-merohedrally twinned crystals of rubredoxin oxygen oxidoreductase from Desulfovibrio gigas

Abstract

Crystals of rubredoxin oxygen oxidoreductase have been obtained and characterized. They belong to space group P2(1)2(1)2, with unit-cell dimensions a = 88.24 (15), b = 101.25 (7), c = 90.80 (3) A. The homodimer (86 kDa) in the asymmetric unit is related by a non-crystallographic twofold rotation axis parallel to the ab 'diagonal' direction, as shown by the self-rotation maximum in the section with chi = 180 degrees. This pseudo-crystallographic symmetry element was also found to be the twinning axis of pseudo-merohedrally twinned crystals, leading to apparent pseudo-tetragonal P42(1)2 crystal symmetry.

Detecting and overcoming hemihedral twinning during the MIR structure determination of Rna1p

“…7c) or if the NCS axis was not highly parallel to the twin operator twofold axis, the inter-domain interactions would differ drastically from the intra-domain crystal contacts and twinned crystal growth would not be possible. A combination of NCS almost parallel to the twin operator has been reported remarkably often for structures from merohedrally twinned protein crystals (Rees & Lipscomb, 1980;Forst et al, 1998;Hillig et al, 1999;Frazã o et al, 1999;Declercq & Evrard, 2001;Larsen et al, 2002;Xu et al, 2003;Lee et al, 2003;Barends & Dijkstra, 2003;Yuan et al, 2003;Golinelli-Pimpaneau, 2005). For crystals of Rna1p, we now see that this NCS nearly parallel to the twin operator was a necessary prerequisite for twinning to happen.…”

“…an orthorhombic space group with a = b). Structure determinations from such pseudo-merohedrally twinned crystals have been described (Ito et al, 1995;Ban et al, 1999;Mueller et al, 1999;Frazã o et al, 1999;Weber et al, 1999;Yang et al, 2000;Declercq & Evrard, 2001;Larsen et al, 2002;Barends & Dijkstra, 2003;Houborg et al, 2003;Dauter, 2003;Rudolph et al, 2004;Hamburger et al, 2004;Golinelli-Pimpaneau, 2005;Lehtiö et al, 2005).…”

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“…The di-iron center and the FMN are located more than 35 Å apart. The homodimer shows a head-totail arrangement through a noncrystallographic two-fold axis 10 , which brings the di-iron site of one monomer close to the FMN of the other monomer (∼6 Å apart). The intradimer contacts involve 12% of an otherwise solvent accessible area extending through one helix of the flavodoxin-like domains plus the region around the cofactors (Fig.…”

“…Aerobically purified native ROO 3 was crystallized by vapor diffusion in a cold room (4-10 °C) to avoid pseudomerohedric crystal twins 10 . Sitting drops of 3 µl of precipitant solution and 3 µl of protein solution (10 mg ml -1 protein) were deposited on microbridges and equilibrated against the precipitant solution (10% (v/v) PEG 6K, 100 mM Tris-maleic acid, pH 6.0).…”

“…Two diffraction data sets were collected, a four-wavelength MAD data set to 2.7 Å resolution at ESRF beamline BM14 and a single wavelength set to 2.5 Å resolution at DESY, EMBL Hamburg outstation, beamline X11 (ref. 10). Diffraction images were processed with HKL 25 .…”