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Figure 3
Interaction studies of hDHTKD1 and hDLST. (a) Constructs of hDHTKD1 and hDLST (black bars) used in the affinity pulldown experiments. (b)–(d) SDS–PAGE showing affinity pulldown of untagged hDLST68–453 from (b) His6-tagged hDHTKD145–919, (c) hDHTKD11–919 and (d) hDHTKD123–919. The original uncropped SDS–PAGE gels are shown in Fig. S7. For panel (b), the lanes loaded are: 1, flow-through; 2–6, wash fractions of increasing imidazole concentration; and 7–11, elution fractions with 250 mM imidazole. SDS–PAGE gel fragments from panels (c) and (d) show only the first three elution fractions i.e. lanes 7, 8 and 9 [equivalent to the lanes marked in red in panel (b)]. (e) Chromatogram and SDS–PAGE from SEC runs of hDHTKD145–919 protein alone (dashed line) and of hDHTKD145–919 co-expressed with hDLST68–453. Elution volumes for the complex peak and hDHTKD1-alone peak are shown. (f) An EM map of the hDLST 24-mer catalytic core overlaid with a humanized model of E. coli DLST (PDB code 1scz). The three views show how the trimer building block (monomers shown as blue, orange and green ribbons) is assembled into the 24-mer core via fourfold (left), twofold (middle) and threefold (right) symmetry axes, respectively. Inset of the left view shows how the first residue (aa 219, red spheres) from each of the 24 hDLST catalytic cores is distributed at the surface of the cube structure.

IUCrJ
Volume 7| Part 4| July 2020| Pages 693-706
ISSN: 2052-2525
https://doi.org/10.1107/S205225252000696X