6112 Nucleic Acids Research, 2008, Vol. 36, No. 19 diﬀerent DNA duplexes. However, crystals with good dif- fraction patterns were obtained only for the inactive var- iant and an 11-mer duplex with the PspGI recognition sequence at its center and complimentary single nucleotide overhangs at its ends (Figure 1). In the same crystalliza- tion conditions, two diﬀerent crystal forms appeared. One crystal form was unsuitable for crystallographic analysis due to perfect merohedral twinning. The other form was rare and could only be grown with the selenomethionine variant of the protein, but proved much easier to handle. In the following, we focus on this crystal form. The PspGI dimer-DNA oligoduplex in the asymmetric unit The asymmetric unit of the non-twinned crystal form con- tains two PspGI protomers and one undecamer duplex. A single (local, non-crystallographic) 2-fold axis relates the two protein subunits and the inner and outer C/G pairs of the PspGI recognition sequence. We therefore conclude that the crystallographic dimer is also the physiologically relevant dimer. As expected, the PspGI subunits assemble similarly as those Ecl18kI subunits that form the func- tional dimer [Figure 2, (11)]. In solution, the two subunits of the PspGI dimer are equivalent, and therefore there is only one way for the duplex to bind, even though the two DNA strands of the duplex diﬀer at the center of the recognition sequence and in the non-recognized ﬂanks of the duplex. In the crystal, diﬀerent environments make the two PspGI subunits non-equivalent, creating two possible binding modes for the DNA duplex. The electron density for DNA bases that do not follow the 2-fold symmetry depends on whether or not binding is inﬂuenced by the crystal environment. This inﬂuence was minimal in the previous Ecl18kI–DNA co-crystal structure and bases appeared averaged (11). In contrast, a single binding mode predominates in the PspGI-DNA co-crystal struc- ture. It stacks the cytosine at the 50-end of the shorter ﬂank against the guanidino group of Arg118 of a neigh- boring molecule. Due to the asymmetry of the ﬂanks of the DNA duplex (Figure 1), the alternative binding mode would lead to a clash instead of this favorable interaction. Gross PspGI structure PspGI has the typical restriction endonuclease fold that is built around a mixed b-sheet with extensive helical Figure 2. A comparison of PspGI-DNA and Ecl18kI-DNA complex structures. (A) PspGI–DNA complex. The two PspGI subunits are shown in ribbon representation in dark and light gray. The DNA is represented by a smoothed backbone and sticks for the bases in yellow, except for the ﬂipped bases, which are shown in orange in all atom representation. (B) Ecl18kI-DNA complex (11). Only a functional dimer is shown. The color coding is analogous to (A), except that the DNA backbone and non-ﬂipped bases are presented in light green color and the ﬂipped bases are shown in dark green color. The helices that are conserved between the two enzymes are numbered (the numbering published previously for Ecl18kI is used). (C) Structure-based alignment of PspGI and Ecl18kI. Conserved helices are numbered consistently in all panels. Stars indicate the active site residues: Glu105, Asp138 (mutated to Ala), Lys160 and Glu173, dots mark the residues involved in sequence recognition (Gln94, Arg164, Glu165 and Arg166). Inverted triangles point to the residues forming the ﬂipped base binding pockets. The length of the unaligned fragments is stated between the arrows.