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. Author manuscript; available in PMC: 2017 Nov 1.
Published in final edited form as: Nat Struct Mol Biol. 2017 May 1;24(6):507–514. doi: 10.1038/nsmb.3407

Figure 2. Engineered disulfides drive ecCTPS assembly and inhibit activity.

Figure 2

a, Design of the ecCTPSCC construct, showing the locations of cysteine mutations at F281 and T335 in the linker-linker and GAT-GAT interfaces, respectively. b, SDS-PAGE gel of ecCTPS WT and CC construct under reducing and non-reducing conditions. Under non-reducing conditions crosslinks are introduced between protomers resulting in dimers (2), trimers (3), and tetramers (4). c, Negative stain images of apo ecCTPSCC under reducing (+DTT) and non-reducing (-DTT) conditions. d, CryoEM reconstruction of ecCTPSCC at 5.7 Å resolution. e, The activity of ecCTPS WT and CC constructs were measured under reducing (+DTT) and non-reducing (-DTT) conditions, using either glutamine or ammonium phosphate (+NH4) as substrates. Values are averages of triplicate experiments +/- s.d. f, The active site cryoEM density from filament structures with no nucleotides present (apo), with products soaked in to pre-formed filaments, or substrates soaked in to pre-formed filaments. Clear density can be seen for products, while there is no density in the overlapping CTP and UTP site when substrates are added, suggesting the filament conformation is intrinsically inhibited.