saturation. Crystals could be ﬂash-cryocooled in the crys- tallization buﬀer (10 ml) mixed with 2 ml of 70% glycerol. First crystal form All crystals of PspGI-D138A with natural methionines and most with selenomethionines belonged to the same crystal form. The best specimens diﬀracted to 1.65 A˚ resolution, with a seemingly ‘clean’ diﬀraction pattern. Nevertheless, experimental phasing and molecular replace- ment with search models based on either the Ecl18kI- DNA complex structure (11) or the EcoRII apo-structure (10) did not succeed. In retrospect, this failure can be attributed to perfect merohedral twinning. As the twin- ning problem greatly complicates the interpretation of the data and degrades the ‘eﬀective’ resolution, this crys- tal form will not be discussed further. Second crystal form A single crystal of the PspGI-D138A selenomethionine variant appeared morphologically distinct from all other crystals. Crystallographic analysis showed that this crystal represented indeed a diﬀerent crystal form that did not suﬀer from any twinning problems. Therefore, the com- plete crystallographic analysis reported in this work was based on this one crystal, which belonged to space group P212121 with cell constants 46.9 A˚, 96.1 A˚ and 127.1 A˚. It contained a PspGI dimer in complex with the 11-mer oli- gonucleotide duplex in the asymmetric unit and diﬀracted to 1.7 A˚ resolution at the beamline BW6 of the DORIS ring at the Deutsches Elektronensynchrotron (DESY) in Hamburg. In order to solve the structure by the multiple anomalous diﬀraction method (MAD), we collected data- sets at a high energy remote wavelength (0.976 A˚) and at the inﬂection point (0.9793 A˚ ). The SHELXC program (17) indicated that the anomalous signals in the two data- sets were over 60% correlated in the lowest resolution shell and over 30% correlated in the shell around 3.0 A˚ , suggesting that the anomalous diﬀerences should be read- ily interpretable. Based on the known amino acid sequence of PspGI-DNA, we expected either four or six selenium sites, depending on whether or not the initiator methio- nines were present and ordered in the crystals. Consistent with this expectation, the SHELXD program (17) readily identiﬁed four sites with signiﬁcant occupancies (1.00, 0.87, 0.39 and 0.38), which stood out against the highest noise signal with an occupancy of 0.08. The correct hand- edness of the selenium substructure was determined by comparing quality parameters after 20 cycles of density modiﬁcation with the SHELXE program (18). The con- trast, a SHELXE measure of map quality, was 0.49 for the correct hand and only 0.31 for the incorrect hand. Although the strongly solvent ﬂattened phases for the correct hand were essentially correct, they were of insuﬃ- cient quality for automatic density improvement and model building by ARP/wARP (19). Inspection of the heavy atom sites suggested that the high- and low- occupancy sites were related by 2-fold non-crystallo- graphic symmetry (NCS). Moreover, the direction of the putative 2-fold axis was supported by a peak of the self- rotation function. As pairs of atom coordinates are Nucleic Acids Research, 2008, Vol. 36, No. 19 6111 insuﬃcient to derive a general NCS symmetry, we supple- mented the two pairs of atom coordinates with the mid- point of the line connecting the two strongest sites, and assumed that this point should be a ﬁxed point of the NCS operation. We then subjected a weakly modiﬁed SHELXE density (only three cycles of density modiﬁcation by this program) to combined solvent ﬂattening and averaging using the program DM. Averaging masks were obtained by automasking, and reﬁnement of NCS operators was allowed to maximize density correlation. The resulting phases to 1.7 A˚ resolution were of suﬃcient quality for ARP/wARP to automatically build a model with alto- gether 509 residues, or 98.5% of the residues in the ﬁnal structure submitted to the PDB. A model of B-DNA with the correct sequence was generated with the program 3DNA (20) and manually modiﬁed to ﬁt the ARP/ wARP generated density. Manual model building and modiﬁcation was done with the programs O (21) and XtalView (22), and reﬁnement was performed with REFMAC (23) and CNS (24). The resulting quality fac- tors of the model appear satisfactory for a structure of 1.7 A˚ resolution (Table 1). Note that the diﬀraction data were collected for the selenomethionine variant of PspGI and that therefore all methionine residues are replaced by selenomethionines in the model. The ﬁnal coordinates and corresponding structure factors were submitted to PDB with the accession code 3BM3. RESULTS Crystallization of the PspGI-DNA complex Wild-type PspGI and an inactive variant of the enzyme, in which the predicted active site residue Asp138 was replaced with alanine, were co-crystallized with several Table 1. Data collection and reﬁnement statistics Data collection statistics Space group a (A˚ ) b (A˚ ) c (A˚ ) Wavelength (A˚ ) Total reﬂections Unique reﬂections Resolution range (A˚ ) Completeness (%) (last shell) I/s (last shell) R(sym) (%) (last shell) B(iso) from Wilson (A˚ 2) Reﬁnement statistics Resolution range (A˚ ) Reﬂections work/test Protein atoms (excluding H) DNA atoms (excluding H) Solvent atoms (excluding H) R-factor (%) R-free (%) Rmsd bond lengths (A˚ ) Rmsd angles (8) Ramachandran core region (%) Ramachandran allowed region (%) Ramachandran additionally allowed region (%) Ramachandran disallowed region (%) P212121 46.9 96.1 127.1 0.976 207295 63138 20.0–1.7 98.7 (98.0) 21.3 (3.2) 6.5 (25.4) 20.7 20.0–1.7 59905/3230 4552 445 306 17.0 19.2 0.013 1.4 94.7 4.4 0.8 0.0