decorations on either side. As in Ecl18kI, there is a long helical region at the N-terminus. The overall structural similarity of PspGI to Ecl18kI is readily apparent, but there are also notable structural diﬀerences, especially at the ends of the sequence and in loop regions (Figure 2). Instead of the long N-terminal a-helix 1 in Ecl18kI, PspGI has a shorter, strand-like irregular structure, a sharp kink and a 310 helix. At the C-terminus, full- length PspGI is 31 residues shorter than Ecl18kI. Mass spectrometry indicates that actual PspGI preparations are further truncated by several residues (data not shown). As a result, PspGI lacks (ordered) equiva- lents for the last two helices of Ecl18kI. PspGI occurs naturally in the hyperthermophile Pyrococcus GI-H, which grows at 858C, while Ecl18kI is produced by the mesophilic Enterobacter cloaceae 18k, which lives at 378C. Therefore, one might expect to ﬁnd generally tighter loops in PspGI than in Ecl18kI, but this is only true in two cases. In between helices 4 and 6, PspGI lacks a connect- ing 310 helix and the a-helix 5, which makes PspGI more compact than Ecl18kI in the region most distant to the catalytic core. Moreover, the region between b-strands 1 and 2 is two residues shorter and better ordered in PspGI. Contrary to expectations, many PspGI loops are longer than their Ecl18kI counterparts. PspGI has additional residues in the region around Ser46 (seven additional resi- dues), an extra winding of helix 8, a longer loop immedi- ately following it (ﬁve additional residues) and a longer helix 10 plus an additional helix (14 additional residues; Figure 2). Active site architecture As we could not obtain diﬀracting crystals of wild-type PspGI with DNA, a variant of PspGI with alanine replacing the predicted active site residue Asp138 was used for the structural studies. In the crystals of this PspGI mutant with DNA, there are no metal ions, and the conformation of the other putative metal binding resi- dues is distorted. Nevertheless, protein backbone super- positions of PspGI, Ecl18kI (11) and the related restriction endonuclease NgoMIV (25) can be used to con- ﬁrm that the PspGI active site residues are Glu105, Asp138, Lys160 and Glu173 as predicted (7). The compar- ison further indicates that in a hypothetical complex of wild-type PspGI with two Mg2+ ions, Asp138 (the residue mutated to alanine in our structure) must serve as a brid- ging ligand; whereas, Glu105 and Glu173 coordinate one metal ion each. In the actual PspGI-D138A co-crystal structure with DNA, Asp138 is mutated and the rotamer conformations of Glu105 and Glu173 are not correct for metal binding. PspGI Lys160 is spatially equivalent to Ecl18kI Lys182 and NgoMIV Lys187 and must therefore be the active site lysine (Supplementary Figure S1). Note that in the Ecl18kI/PspGI/EcoRII group of restriction endonucleases, the active sites are fairly distant from the central nucleotides of the target pseudopalindrome, so that the active site mutation and the lack of metal ions are unlikely to have inﬂuence on the interaction of PspGI with the central bases. Nucleic Acids Research, 2008, Vol. 36, No. 19 6113 Overall architecture of the bound DNA The conformation of the DNA duplex in the complexes with PspGI and Ecl18kI is generally similar. In both cases, the bases at the center of the recognition sequence are extruded from the DNA base stack and ﬂipped into extra- helical positions. Unlike many other nucleotide ﬂipping enzymes, which are not restriction endonucleases (26–29), PspGI and Ecl18kI ﬂip nucleotides of both DNA strands and do not insert any amino acid residue into the DNA stack to replace the missing bases, at least not permanently. As there is no intercalation to keep bases adjacent to the ﬂipped bases apart, these come closer towards each other than 6.8 A˚, which would be the normal spacing between them in regular B-DNA. Moreover, PspGI and Ecl18kI kink the DNA so that the major groove gets compressed and the minor groove expanded. Despite these similarities, ﬁne diﬀerences (mostly in dihedral angles) result in slightly diﬀerent posi- tions of the ﬂipped bases: in the PspGI co-crystal struc- ture, the ﬂipped bases are displaced slightly more toward the 50-ends of their DNA strands than in the Ecl18kI co-crystal structure (Figure 3). It is not clear whether the two complexes map out diﬀerent positions along the tra- jectory of the base during ﬂipping, or whether they repre- sent genuinely diﬀerent endpoints of ﬂipping in the diﬀerent enzymes, and, if the latter is true, whether this diﬀerence might account for their diﬀerent speciﬁcities. DNA distortions that lead to nucleotide flipping In the crystal structure of Ecl18kI with DNA, some mechanistic details of nucleotide ﬂipping were obscured by the coexistence of two DNA binding modes, which blurred the electron density in the key regions. In contrast, the PspGI-DNA crystal structure is largely free from this complication, allowing us to discuss nucleotide ﬂipping in more detail (Figure 4). Independent reﬁnement of the two DNA strands of the PspGI-DNA co-crystal structure shows that the dihedral angles w, which describe the rota- tion around the glycosidic bond, are radically diﬀerent between the two ﬂipped nucleotides. The ﬂipped T-nucleotide is present in the usual anti conformation, but the A-nucleotide assumes the normally disfavored syn conformation. All other backbone torsion angles are roughly similar for the two strands. Qualitatively, only the ﬂipped nucleotides and their immediate neighbors appear to diﬀer signiﬁcantly in shape and position from their counterparts in regular B-DNA. Quantitatively, a com- parison of actual dihedral angles in the crystal structures with values for idealized B-DNA shows that major diﬀer- ences are limited to nucleotides 2 to +1. For the ﬂipped nucleotides, the most conspicuous feature is the syn con- formation of the ﬂipped adenine, but the dihedral angles a and g diﬀer radically and the z angles to a lesser extent from the values that would be expected for regular B-DNA. For the 2 and 1 nucleotides, the biggest dif- ferences are found for the dihedral z angles, and for the +1 nucleotides, the most unusual angles are the g angles. The comparison of DNA distortions in the PspGI-DNA and Ecl18kI-DNA co-crystal structures is complicated by the need for double-conformation reﬁnement in the case