belongs to the space group P3221, and diﬀracted to better than 1.6 A˚ at beamline 3W1A of Beijing Synchrotron Radiation Facilities (BSRF). Diﬀraction data were collected on a MAR165 CCD camera (MARresearch GmbH, Hamburg). The wavelength was set to 1.24 A˚ , at the higher energy side of the absorption peak of zinc. The data with anomalous signals were processed using the program DENZO and SCALEPACK (35). The statistics for the X-ray data collection and processing of the P3221 crystal form are summarized in Table 1. The zinc site was located using the program SHELXD (36) and the heavy atom parameters were reﬁned using the program SOLVE (37). The initial phases were obtained by the program OASIS-2004 (38), then reﬁned by using the program DM (CCP4) (39). Finally, ARP/warp (40) was used for the model auto-building. Since the initial phases and auto-traced model were of good qualities, the subsequent reﬁnement work could be easily carried out by using the program CNS (41). Manual adjustment of the structure was done with the program O (42), and a succinic acid as well as water molecules were added into the positive diﬀerence densities. The ﬁnal model was reﬁned by REFMAC5 (43). Mutant design and site-directed mutagenesis The design of mutants was based on a DNA–protein complex model between the W-box with the sequence 50-ATCGTTGACCGAGTTGA-30 and AtWRKY1-C, which was generated from the known GCM–DNA complex (44) with the PDB ID 1ODH. GCM in the complex was replaced by AtWRKY1-C with least square (LSQ) ﬁtting between AtWRKY1-C and GCM using the program O (42). The root mean-square standard devia- tions (RMSD) after LSQ was 2.3 A˚ for 68 Ca atoms. A B-DNA model for speciﬁc binding was prepared using the program CNS (41) and NAB (45) with the sequence 50-ATCGTTGACCGAGTTGA-30. DNA in the GCM–DNA complex was then replaced using the func- tion of LSQ ﬁt in XtalView (46). The wild-type AtWRKY1-C cDNA was used as a template for the mutagenesis primers. Site-directed mutagenesis by PCR was introduced into AtWRKY1-C. DNA sequencing veriﬁed the introduction of the desired mutations and demonstrated that no unwanted mutations were present in the mutated protein sequences. Electrophoretic mobility shift assays (EMSA) The oligonucleotides 50-ATCGTTGACCGAGTTGA-30 and 50-TCAACTCGGTCAACGAT-30 containing the optimal binding site for AtWRKY1 were annealed to form DNA duplex. The typical binding reaction (20 ml) contained 3 mg dsDNA, 20 mM HEPES/KOH (pH 7.2), 40 mM KCl, 1 mM EDTA, 0.5 mM DTT, 10% glycerol and 5 mg puriﬁed wild-type AtWRKY1-C protein or mutant proteins. The binding reaction mixture was incubated at room temperature for 20 min and the complex was separated from the free duplex on a 12% non-denaturing polyacrylamide gel electrophoresis (PAGE) in 0.5 TBE at 70 V for 3.5 h. The gel was stained with EB. Images were captured using the Nucleic Acids Research, 2007, Vol. 35, No. 4 1147 Table 1. Data collection, phasing and reﬁnement statistics (SAD) Data collection Reﬁnement Space group P3221 Resolution (A˚ ) Cell dimensions No. of reﬂections a, b, c (A˚ ) 45.55,45.55,68.96 Rwork/Rfree (%) a, b, g (8) 90,90,120 No. of atoms Resolution (A˚ ) 30–1.6 (1.66–1.6) Protein Rsym or Rmerge (%) 9.1 (35.4) Ligand/ion I/ I 22.9 (2.9) Water Completeness (%) 97.9 (95.5) Average B-factors Redundancy 5.6 (4.6) RMSDa bond lengths (A˚ ) RMSDa bond angles (8) 30–1.6 10 536 17.9/20.4 727 609 8/1 100 20.95 0.015 1.61 aRMSD in bond lengths and angles are the root-mean squared (RMS) deviations from ideal values. ﬂuorescence imaging system (Alpha Innotech Corporation, USA). A more ‘standard’ EMSA analysis was also performed. The standard binding reaction (20 ml) contained 0.5 mg of poly(dI-dC), 20 mM HEPES/KOH (pH 7.2), 40 mM KCl, 1 mM EDTA, 0.5 mM DTT, 10% glycerol, 0–180 ng puriﬁed proteins were added and 10 ng labeled double-stranded synthetic oligonucleotide, labeled with [g-32P]ATP using T4 polynucleotide kinase (NEB). For the competition assay, 100-fold molar excess of the unlabeled speciﬁc probe was added. DNA–protein complexes were allowed to form at room temperature for 30 min and then resolved on a 12% non-denaturing polyacrylamide gel in 0.5 TBE. The gel was dried and exposed for 2 h on X-ray ﬁlms. CD spectroscopic assays All the CD spectra of the wild-type and mutant AtWRKY1-C proteins were recorded on a Jobin Yvon CD 6 spectrometer (Longjumeau, France) at 298 K. The CD spectra of all proteins were recorded in phosphate buﬀer saline (PBS) buﬀer, pH 7.5. For near-UV–CD spectrum, a cell with a path length of 1 mm was used. Each spectrum was the average of four scans corrected by subtracting a spectrum of the buﬀer solution in the absence of proteins recorded under identical condition. Each scan in the range of 195–260 nm was obtained by taking data points every 0.5 nm with a 2-nm bandwidth and integration time of 1 s. RESULTS AND DISCUSSION AtWRKY1 is involved in the SA signaling SA is an important signal molecule in plant disease resistance. It has been previously shown that SA treatment can induce the expression of PR genes and activate systemic acquired resistance (SAR), which makes plants resistant against a spectrum of pathogens (47). To study the potential involvement of AtWRKY1 in SA signaling pathway and its possible relation to NPR1, a key protein in SAR, we characterized the expression proﬁle of AtWRKY1 by RNA gel blot in wild-type Arabidopsis seedlings, npr1-3 mutant (12) and NPR1-H line (33) in