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. 2008 Nov 1;64(Pt 11):1034-8.
doi: 10.1107/S1744309108031722. Epub 2008 Oct 28.

Crystallization of Doc and the Phd-Doc toxin-antitoxin complex

Abel Garcia-Pino  1 , Minh-Hoa Dao-Thi, Ehud Gazit, Roy David Magnuson, Lode Wyns, Remy Loris
Affiliations

Crystallization of Doc and the Phd-Doc toxin-antitoxin complex

Abel Garcia-Pino et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. .

Abstract

The phd/doc addiction system is responsible for the stable inheritance of lysogenic bacteriophage P1 in its plasmidic form in Escherichia coli and is the archetype of a family of bacterial toxin-antitoxin modules. The His66Tyr mutant of Doc (Doc(H66Y)) was crystallized in space group P2(1), with unit-cell parameters a = 53.1, b = 198.0, c = 54.1 A, beta = 93.0 degrees . These crystals diffracted to 2.5 A resolution and probably contained four dimers of Doc in the asymmetric unit. Doc(H66Y) in complex with a 22-amino-acid C-terminal peptide of Phd (Phd(52-73Se)) was crystallized in space group C2, with unit-cell parameters a = 111.1, b = 38.6, c = 63.3 A, beta = 99.3 degrees , and diffracted to 1.9 A resolution. Crystals of the complete wild-type Phd-Doc complex belonged to space group P3(1)21 or P3(2)21, had an elongated unit cell with dimensions a = b = 48.9, c = 354.9 A and diffracted to 2.4 A resolution using synchrotron radiation.

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Figures

Figure 1
Figure 1
Purification of DocH66Y. (a) Coomassie-stained SDS–PAGE of the affinity chromatography fractions. Lane 1, molecular-weight markers (kDa); lane 2, soluble protein before induction; lane 3, soluble protein 2 h after induction; lane 4, DocH66Y after purification on the His-trap affinity column. (b) Identification of DocH66Y on a Western blot developed with rabbit antibodies directed against wild-type Doc. The lanes are equivalent to those in (a).
Figure 2
Figure 2
Different crystals of free DocH66Y and its complexes with Phd. (a) Initial crystals obtained of free DocH66Y. (b) Large crystals of free DocH66Y obtained after seeding. (c) Monoclinic crystals of DocH66Y–Phd52-73Se. (d) Hexagonal crystal form of the Phd–Doc complex. The scale bar corresponds to 0.1 mm.
Figure 3
Figure 3
Typical diffraction patterns of (a) DocH66Y–Phd52-73Se and (b) Phd–Doc. In both cases the rotation angle is 1°. The inset in (b) shows a clear separation of the reflections in the c* direction. (c) Self-rotation function with κ = 180° for DocH66Y crystals. Red circles indicate peaks corresponding to noncrystallographic symmetry axes.
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