Extended Data Fig. 6: The crystal structure of apo GiGet3.
A) Size exclusion chromatograms of nickel eluate for Get3 and (B) SDS-PAGE gels of the peak highlighted in (A). GiGet3 was purified 10-20 separate times in order to produce enough material for crystal trays and cryo-EM analysis. B) The asymmetric unit of apo GiGet3 contains two distinct monomers of Get3 (gray), which are designated apo1 and apo2. Each monomer pairs with its respective symmetry equivalent in the neighboring asymmetric unit to form two distinct dimers. One dimer, apo1 (magenta), has the ’open’ conformation. The other dimer, apo2 (purple), has a conformation that is slightly more closed than the previously seen ‘open’ conformations. C) Alignment of the two monomers highlighting additional slight differences between. Apo1 is colored from N to C terminus using the viridis color map and apo2 in grey. The monomers are aligned to the P-loops and there are no notable differences in the NBD. The flexibility of the CBD is demonstrated here. Most distinct is a shift in the loop between H5 and H6 (residues 121-139) with most of this region disordered in apo2. Additionally, H5 is shorter at the N-terminus in apo2 compared to apo1 due to a close contact with a symmetry mate that presumably disrupts the helix (red arrows in F). A 2Fo-Fc map shown as blue mesh at 1.0 sigma showing the region that includes the Zn2+ ions on the symmetry axes for (D) apo1 (outlined in cyan) and (E) apo2 (outlined in orange); these positions correspond to the arrows in (B). F) A single layer of the crystal lattice for apo GiGet3 crystals. Grey monomers define the asymmetric unit and symmetry related dimers are colored for either apo1 (magenta) or apo2 (purple).
