Supplementary Figure 2: SYCP1 is a parallel tetramer that bifurcates into two C-terminal coiled-coil dimers.

(a) Far UV CD spectra and (b) CD thermal denaturation of SYCP1 αCore-ΔNtip (112-783) (black), αN-ΔNtip (112-362) (grey), αN-tetramer (206-362) (narrow dashes) and αC-dimer (358-783) (wide dashes). Helical content was estimated (as indicated) through deconvolution of spectra with data fitted at normalised rmsd values of 0.005, 0.009, 0.003 and 0.003 respectively. Thermal denaturation was recorded as % unfolded based on the helical signal at 222 nm; melting temperatures were estimated at 52 °C, 38 °C, 38 °C and 37 °C respectively. (c-f) SEC-SAXS analysis of SYCP1 αCore-ΔNtip, αN-tetramer and αC-dimer, and their N-terminal MBP fusions. (c-d) Scattering intensity plots with fits used for P(r) distributions shown as black lines. (e) Guinier analysis to determine the radius of gyration (Rg) with linear fits shown in black. Q.Rg values were < 1.3. The Guinier regions of αCore-ΔNtip and αC-dimer were too narrow for analysis; their Rg values were calculated as 254 Å and 196 Å from real space P(r) distributions. (f) Guinier analysis to determine the radius of gyration of the cross-section (Rc), with the linear fits highlighted in black. Q.Rc values were < 1.3. (g-j) SEC-MALS analysis of SYCP1 fusion proteins; light scattering (LS) and differential refractive index (dRI) are shown as solid and dashed lines respectively, with fitted molecular weights (Mw) plotted as diamonds across elution peaks. (g) MBP-αCore-ΔNtip (black) is a 477 kDa tetramer (theoretical tetramer - 499 kDa) whilst MBP-αC-dimer (grey) is a 185 kDa dimer (theoretical dimer - 191 kDa). (h) MBP-αN-tetramer (black) is a 232 kDa tetramer (theoretical tetramer - 255 kDa) whilst MBP (grey) is a 43 kDa monomer (theoretical monomer - 45 kDa). (i) RecE-αN-tetramer (black) and RecE (grey) are tetramers of 208 kDa (theoretical tetramer - 214 kDa) and 132 kDa (theoretical tetramer - 136 kDa) respectively. (j) GST-αN-tetramer (black, right) is a 194 kDa tetramer (theoretical tetramer - 195 kDa), whilst GST-αC-dimer (black, left) and GST (grey) are dimers of 157 kDa (theoretical dimer - 160 kDa) and 58 kDa (theoretical dimer - 59 kDa) respectively. (k-n) SEC-SAXS analysis of GST fusions of αN-tetramer and αC-dimer. (k) Scattering intensity plots. (l-m) Guinier analysis to determine the radius of gyration (Rg). (n) P(r) distributions of GST-αN-tetramer (black), GST-αC-dimer (wide dashes) and GST (grey) showing maximum dimensions of 282 Å, 677 Å and 99 Å respectively. In GST-αN-tetramer, the presence of short inter-atomic distance peaks, and the absence of peaks close to Dmax values, is consistent with the relative orientation of GST molecules within parallel coiled-coil structures. Peaks corresponding to intra-GST and inter-GST distances are indicated.