1726 Nucleic Acids Research, 2010, Vol. 38, No. 5 analysed by SDS–PAGE and those containing ArdB were pooled and concentrated. Puriﬁed ArdB/KlcA was stored at 20 C in buﬀer A supplemented with glycerol to 50% (v/v). Analysis of proteins The methods employed for assaying ardB activity in vivo used the eﬃciency of plating (eop) of phage lambda (virulent), v, on the various strains of E. coli as described previously in detail (8,13). The NM and NK E. coli strains were transformed with the derivatives of pET24a or pET20b described above but protein overexpression was not induced. Instead, we relied upon the small amount of leaky expression to produce the anti-restriction proteins in suﬃcient quantity to demonstrate anti-restriction in vivo. All assays were performed in triplicate and at least 50 phage plaques per plate were counted. The standard deviation on the anti-restriction and anti-modiﬁcation values is ± 25%. To assess the possible role of the ClpXP in the anti-restriction behaviour of ArdB and KlcA, the ability of E. coli NM1041 (hsd+ clpX ) transformed with plasmids expressing ardB or klcA to survive on an agar plate containing 2-aminopurine (2-AP) was assessed (32). The strain will not grow in the presence of 2-AP unless the EcoKI RM system is inactivated by the production of active ArdB or KlcA. A single colony from an agar/ carbenicillin plate of E. coli NM1041 transformed with either pET20b or pET20b-klcA136 was streaked on an agar plate with or without 2-AP and incubated overnight at 37 C. This was repeated in triplicate with diﬀerent colonies. Fourier transform ion cyclotron mass spectrometry (33), isothermal titration calorimetry (10,13,33), size exclusion chromatography (34) and assays of EcoKI endonuclease activity (8,13,34) were performed as described previously. To investigate whether ArdB or KlcA interfered with the assembly of the EcoKI nuclease, 42 nM EcoKI Mtase and 100 nM HsdR proteins (34) were incubated separately with a 20-fold excess of ArdB or KlcA in the nuclease reaction buﬀer for 2 min at 37 C. Equal volumes of these two samples were then mixed and pBRsk1 DNA added to 3 nM. After incubation of this reaction mix for 8 min at 37 C, the reaction was stopped as above. NMR spectroscopy and structure calculations The KlcA protein from B. pertussis was uniformly labelled with 15N and 13C by growing the recombinant strain in M9-minimal medium containing 15NH4SO4 and 13C-D- glucose as the sole nitrogen and carbon sources, respec- tively. The labelled protein was puriﬁed as described above. Samples for NMR measurements contained 0.7 mM KlcA, 90% H2O/10% D2O and 0.05% NaN3 in 20 mM deuterated sodium acetate buﬀer (pH 6.0). All NMR experiments were carried out at 298 K unless stated otherwise on a Bruker AVANCE 800 MHz with a 5-mm triple resonance cryoprobe. NMR experiments for 1H, 15N and 13C backbone and side-chain resonance assignments included: two-dimensional (2D) 1H-1H TOCSY, 1H-1H NOESY, 1H-15N HSQC, 1H-13C HSQC, (HB)CB(CGCD)HD/(HB)CB(CGCDCE)HE, three-dimensional (3D) HNCO, HN(CA)CO, CBCANH, CBCA(CO)NH, HBHA(CO)NH, HBHANH, (H)CCH-TOCSY, H(CCO)NH, C(CO)NH (35). 3D 15N- and 13C-edited NOESY-HSQC spectra were acquired to obtain distance constraints (36,37). Aromatic 1Hd and 1He frequencies were obtained from (HB)CB(CGCD)HD/ (HB)CB(CGCDCE)HE spectra (38) where the aromatic 1H frequencies are correlated with the assigned 13Cb frequencies of the aromatic residue. NMR data were processed using the Azara suite of programs (Wayne Boucher, Department of Biochemistry, University of Cambridge, UK, http://www.ccpn.ac.uk/azara/) and analysed using Ccpnmr Analysis (39). NOE assignments were generated during the initial rounds of structure analysis using CANDID (40) in CYANA 2.1 (41). Dihedral constraints were generated using TALOS (42). The NOE assignments obtained by CANDID were checked manually and from the 100 structures calculated, 20 structures with lowest target function were selected for further analysis. The structures were further reﬁned with the incorporation of residual dipolar couplings (RDCs) using CNS (43). PROCHECK-NMR (44) and WHATIF (45) were used to analyse the quality of the structures. MOLMOL (46) and PYMOL (http://www.pymol.org) were used for visualisation. A 200 mM 15N-labelled KlcA sample in 20 mM deuterated sodium acetate, 0.05% NaN3, pH 6.0 in 90% H2O/10% D2O aligned with 6.25 mg/ml of Pf1 ﬁlamen- tous phage (Pf1 protease and RNase free, Profos) was used to measure RDCs. Magnetic alignment was moni- tored by 1D 2H NMR spectra as a function of phage concentration. A 7 Hz residual quadrapolar splitting of the deuterium signal was achieved with 6.25 mg/ml phage. 1H-15N splittings were measured under isotropic and partially aligned conditions using 2D IPAP 1H-15N HSQC experiments (47). Analysis and back calculation of the RDCs were performed with REDCAT (48). Relaxation experiments were performed on a 600 MHz Bruker Avance spectrometer at 25 C with 200 mM 15N labelled KlcA. For the longitudinal (T1) and transverse (T2) relaxation data for the backbone 15N nuclei of KlcA, water was kept along the z-axis during the entire pulse sequence (49) and extra delays were used in the T1 experiment to eliminate diﬀerential heating arising from the variations of the relaxation delay (50). Eight interleaved spectra were recorded for each relaxation delay T1 (51 twice, 401, 601, 801, 901, 1001 and 1201 ms) and T2 (16 twice, 48, 64, 80, 96, 112 and 144 ms). T1 and T2 cross-peak intensities were measured from peak heights and ﬁt to an exponential decay function I(t) = I(0)e Rt where I(0) and I(t) are the cross-peak inten- sity at time 0 and time t, where time is the relaxation delay and R (1/T) is the relaxation rate (39). For the 2D 1H-15N heteronuclear NOE experiment (49) a 3 s pre-saturation period and a 2 s relaxation delay was used; the control experiment had an equivalent 5 s delay. Spectra from the heteronuclear NOE experiment were extended by linear