a, Reaction scheme for ACLY. In the first step, ACLY undergoes autophosphorylation at His760. Citryl-phosphate (citryl-P) and citryl-CoA form non-covalent enzyme-intermediate complexes. b, Left, representative class averages for hACLY as obtained by negative-stain electron microscopy. The size of the box is 40 × 40 nm. Right, flowchart of the 3D reconstruction in C2 symmetry. Negative-stain electron microscopy analysis was performed on a single sample of purified hACLY (n = 1). SDG, stochastic gradient descent. c, Coomassie-stained SDS–PAGE gel for recombinantly produced ACLY enzymes. Lane 1, hACLY-A/B; lane 2, hACLY; lane 3, M. concilii ACLY-A/B; lane 4, C. limicola ACLY-A/B; lane 5, hACLY(His760Ala). In this study, each protein was purified several times, and the electrophoretic profile of each sample in the gel shown is representative for different protein batches. For gel source data, see Supplementary Fig. 1. d, Size-exclusion chromatography (SEC) elution profile of hACLY-A/B plotted as the light scattering intensity at 90° in function of the elution volume. The reported molecular mass by multiangle laser light scattering (MALLS) represents the average molecular mass ± s.d. across the elution peak. The theoretical mass for hACLY-A/B is 462 kDa. Number of samples for hACLY-A/B analysed: n = 1. e, Reaction rates for hACLY-A/B, hACLY and hACLY(His760Ala) plotted as a function of ATP concentration. For hACLY-A/B and hACLY, data replicates (n = 4, in which n represents a different protein batch) were fitted by a Michaelis–Menten equation and the obtained Michaelis constant (K m ) and turnover number (k cat ) values (mean + s.e.m) are shown. The kinetic parameters for hACLY-A/B and hACLY are significantly different via two-tailed unpaired t-tests: P = 0.0002 (comparing k cat ); and P = 0.0156 (comparing K m ). For the hACLY(His760Ala) mutant, the number of replicate batches: n = 1. f, Representative crystal structure for hACLY-A/B extracted from the P1 crystal form and coloured by chain. Bound substrates are shown as coloured spheres. g, View on the helical bundle core of the CCL module with the protruding two-helix stalk regions indicated. CoA-binding domains are omitted for clarity. h, Overlay of the four hACLY-A/B crystal structures extracted from the P1 and C2 crystal forms. The overlay is based on the superposition of the CCL modules. Structures are coloured according to the scheme in Fig. 1a. A zoom-in view shows the structural plasticity around the two-helix stalk region. i, View on the helical bundle core of CCL from H. thermophilus coloured by chain. The N and C termini of a single chain are indicated. Source data