1730 Nucleic Acids Research, 2010, Vol. 38, No. 5 1H, 15N and 13C chemical shift assignments and structure calculations for KlcA136 The recorded 1H-15N HSQC spectrum, Figure 6, was con- sistent with that of a folded protein exhibiting well dispersed signals and very little spectral overlap. Backbone and side-chain resonances of KlcA136 were assigned based on 2D and 3D triple-resonance experi- ments. Peaks in the 3D 13C and 15N edited NOESY spectra were picked and the NOE cross-peak assignments were obtained from the automated iterative assignment program CANDID that works in conjunction with 3D structure calculations in the program CYANA 2.1. Figure 5. The eﬀect of adding ArdB or KlcA during the assembly of the EcoKI RM enzyme. Where relevant, Mtase and the R subunit were preincubated at 25 C in the presence of a 20 excess of ArdB or KlcA protein for 5 min prior to mixing and addition of DNA. Reactions were performed at 37 C for 8 min. Lane 1, uncut pBRsk1 (3 nM); lane 2, pBRsk1 (3 nM) digested with EcoKI (30 nM); lane 3, pBRsk1 digested with reconstituted nuclease (equivalent to 30 nM EcoKI); lane 4, nuclease reconstituted in the presence of ArdBYAF (20-fold excess over nuclease); lane 5, 20-fold excess ArdBYFJ; lane 6, 20-fold excess KlcA136; lane 7, pBRsk1 in the presence of R subunit only; lane 8, pBRsk1 in the presence of Mtase only. M, 1 kbp size marker. Using NOE restraints, TALOS dihedral restraints and 1H-15N RDCs (Supplementary Figure S1), the lowest energy ensemble of 20 structures of KlcA136 were calculated (Supplementary Figure S2). The calculations converged well, yielding root mean square deviations of 0.63 and 0.65 for backbone and all heavy atoms in structured regions (residues 7–142). Only a subset of RDCs, as speciﬁed in the ‘Methods’ section for the calcu- lation of the rotational diﬀusion tensor, was used in the structure calculations. The structures were validated using PROCHECK and WHATIF. In the Ramachandran plot, 98.5% of residues appeared in the most favourable and additionally allowed regions. WHATIF packing analysis yielded an average quality control z-score of 1.432 sug- gesting a good quality NMR structure. The structure of KlcA136 reveals a rigid globular protein with a novel fold The KlcA136 protein structure is made up of a single domain (73) containing ﬁve a-helices (labelled 1–5), a single 310-helix and ﬁve b-strands (labelled A–E) and belongs to the mixed a/b class of proteins, Figure 7. The ﬁve b-strands form two separate beta-sheets, the ﬁrst composed of anti-parallel b-strands A, B and C and the other made up of anti-parallel b-strands D and E. b-Strand A usually occurs as an isolated b-bridge from the ensemble consensus. The two b-sheets closely ﬂank the C-terminal three a-helices (3–5) on either side. These three helices appear to run in an almost anti-parallel fashion to their helical axes in a classical knobs-in-holes arrangement of a three-helical bundle. Helix 3 is the longest of the helices located at the centre of the fold Figure 6. 2D 1H-15N HSQC spectrum of KlcA136 at 800 MHz.