1732 Nucleic Acids Research, 2010, Vol. 38, No. 5 residues or those exhibiting slow exchange broadening. A subset of 97 residues was used in the determination of the diﬀusion tensor. In this subset the average values of T1 and T2 were 737 and 86 ms, respectively. The relative moments of inertia values were found to be Iz: 1.00, I : y 0.9858 and Ix: 0.8629. Iy and Ix are very close to 1.00, implying that the protein does not deviate signiﬁcantly from a sphere, having slightly oblate shape. Given the nearly spherical shape, an isotropic diﬀusion tensor with a Diso = 1.8 107 s 1 was calculated and the average rotational correlation time (tc) was found to be 8.83 ns ± 0.01, a value expected for a protein of this size. In order to extract microscopic parameters of motion from the relaxation data a model-free approach was used, which assumes the overall and internal motions of the molecule are independent. Based on the above analysis of the relaxation data, an isotropic model was selected. Relaxation data for most residues (73%) were adequately described by the generalized order parameter S2 suggest- ing a rigid molecule overall. The order parameter is a measure of the degree of spatial motion of the N–H bond where a value of zero means that the bond vector has unrestricted motion while one implies a completely rigid bond. An additional parameter (Rex) was required to ﬁt 17 residues, with residues A14, A16, A91, E107, I108, L117 and Y121 showing signiﬁcant Rex values (>2 Hz), suggesting that these N–H sites experience multiple envi- ronments interconverting on the micro- to millisecond time scale. Eleven residues (E4, M31, V38, W41, E47, Y56, F64, F99, Q103, F123 and A128) did not ﬁt the model. The order parameter S2 as a function of the residue number is shown in Supplementary Figure S3. The distribution of S2 values visualized on the structure shows that the mobility on the nanosecond time scale is slightly restricted in secondary structure elements compared to loops (Supplementary Figure S4). The largest mobility indicated by the smallest S2 values was observed with residues at the N-terminus and with residues between the end of strand C and the start of helix 3. The surface of KlcA136 reveals charged and hydrophobic patches but little sequence conservation for residues in these patches An electrostatic surface representation generated by GRASP shows that the surface of KlcA136 is predominantly negatively charged, consistent with its the- oretical pI (4.76), Figure 8. The negatively charged side-chain residues on the KlcA136 surface reveals two distinct clusters (i) an ‘Aspartate cluster’ made up of ﬁve linearly arranged residues i.e. D61 (in the loop between b-strands B and C), D110, D112, D115, D119 (in the loop between a-helix 3 and 4 and on a-helix 4) and (ii) a ‘Glutamate cluster’ that contains amino acid residues E47 (in the loop between a-helix 2 and b-strand B), E75 and E77 (on b-strand D), E86 (on b-strand E) and E133 (on a-helix 5). In addition to the ﬁve residues present in the ‘Glutamate cluster’ are residues E15, D69 and D90, which are also located nearby in 3D space. A striking linear arrangement of positive charge involving arginine side-chains is also evident, predominantly on the opposite side of the KlcA136 protein surface with respect to its neg- atively charged clusters. This ‘Arginine cluster’ contains seven residues: R29, R33, R43, R44 on a-helix 2, R48 in the loop between a-helix 2 and b-strand B, R84 on b-strand E and R120 on a-helix 4. Even though three distinct regions of charge are apparent from the electrostatic surface representations, only a handful of the charged residues are conserved in the 43 non-redundant sequences identiﬁed (Supplementary Figure S5). These include R18, E35, D61, D90, R125, E133 and D142. Figure 8. Electrostatic surface representation of the KlcA136 protein. Two views rotated by 180 about the y-axis of a GRASP electrostatic surface representation of the KlcA136 protein. The molecule appears to expose many charged residues, labelled. Negative charge is coloured red and positive charge coloured blue, ranging from 10 kBT to +10 kBT (kB: Boltzmann constant; T: temperature in Kelvin).