Figure 2. The structure of HiSiaQM.

(a) Coulomb maps for the parallel (3.36 Å) and antiparallel (2.99 Å) HiSiaQM homodimers. The periplasmic surfaces of the monomers are facing the same direction for the parallel dimer (PDB: 8THI), whereas the periplasmic surface of one monomer is rotated 180° for the antiparallel dimer (PDB: 8THJ). The transport domain (orange and gold) is in the ‘elevator down’ conformation in all four monomers. The dimeric interface in both maps is distanced and neither has significant protein–protein interactions. The maps are coloured according to the topology in (c). Density consistent with phospholipids is coloured grey and is particularly present in the dimer interface of the higher resolution antiparallel dimer map. (b) Structural model of the HiSiaQM monomer. The transport domain is in the ‘elevator down’ conformation with the substrate-binding site facing the cytoplasm. (c) The topology of HiSiaQM is the same as the non-fused PpSiaQM with the addition of the fusion helix. The M-subunit forms the transport domain (orange and gold) and bracing arm helices (pink) as well as a large portion of the scaffold (purple and blue). The Q-subunit is entirely used as a scaffold for the elevator transport mechanism. The fusion helix (purple) connects the scaffold and adds to its size. It also forms a short horizontal helix, similar to the arm helices of the M-subunit. (d) A structural overlay of HiSiaQM (2.9 Å structure, green; 4.7 Å structure, purple) and PpSiaQM (2.9 Å structure, orange) shows that the helices of the structures are well aligned, and all three structures are in the same conformation.

Figure 2—figure supplement 1. Size-exclusion chromatography traces of SiaQM transporters suggests that HiSiaQM exists as multiple species in detergent.

(a) HiSiaQM purified in dodecyl-β-d-maltoside (DDM) (green trace) results in significant aggregation at 45–50 mL, which is not apparent when solubilised in lauryl maltose neopentyl glycol (L-MNG) (blue and pink traces). HiSiaQM purified in a low concentration of L-MNG (blue trace) results in a higher order species with a peak at 57 mL compared to HiSiaQM purified in a high concentration of L-MNG (pink trace), which has a peak at 65 mL. At a similar protein concentration (~5 mg/mL) in DDM, HiSiaQM elutes as a single peak between these volumes at 60 mL (green trace). For comparison, non-fused SiaQM from P. profundum expressed with the same purification tag and purified in a low concentration of L-MNG (black trace) has a dominant peak at 67 mL, characteristic of a monomer, and this greatly contrasts against HiSiaQM in low L-MNG (blue trace). (b) SDS-PAGE gel (left) of L-MNG purified HiSiaQM that consistently ran as two bands (~45 and ~75 kDa). Protein of this purity (>95%) in L-MNG was used for all subsequent experiments, including for further reconstitution into amphipol or nanodiscs. Western blot (right) with anti-Xpress primary antibody identifies both large bands that appear on SDS-PAGE following purification to be HiSiaQM. A small amount of a lower molecular weight contaminant or degradation product was also present. (c) SDS-PAGE gel demonstrating the purity of HiSiaP. A single band at ~35 kDa indicates that HiSiaP is greater than 95% pure. Protein of this purity (>95%) was used for all subsequent experiments.

Figure 2—figure supplement 2. Cryo-electron microscopy (cryo-EM) workflow for structure determination.

The two classes were determined as 36% of the particles (antiparallel dimer) and 36% (parallel dimer).

Figure 2—figure supplement 3. Representative density of the helices of HiSiaQM.

Clear side-chain densities are present for almost all of the residues of HiSiaQM. Density for the antiparallel dimer is shown as it is higher resolution and was used for the structural analysis in this work.