SAXS data for homodimeric hCCS were collected on the SWING beamline at SOLEIL synchrotron, St Aubin, France, using the HPLC integrated SAXS setup 211. Protein (400 μg
in 40 μl storage buffer) was loaded onto a pre-equilibrated Shodex KW402.5-4F 150 kDa SEC column at a flow rate of 80 μl/min using an Agilent 1200 series HPLC at 25 oC before
passing through the beam with wavelength 1.03 Å. The sample to detector distance was set to 180 cm. 250 exposures of 2 seconds duration were taken over the course of protein elution with 1 second dead time between each. 20 frames were taken prior to protein elution for buffer subtraction purposes.
SAXS data for the heterodimeric hCCS-SOD1 complex were acquired in a similar fashion however a Superdex 200 16/60 column was used for protein separation. Column temperature was kept at 20 oC using a chilled water bath and a flow rate of 0.75 ml/min
was established. 250 frames were taken over the course of protein elution with a 3 second exposure time separated by 1.5 seconds dead time. 100 frames were taken before the protein eluted for buffer subtraction. Samples of differing concentrations were loaded in a 900 μl volume.
Scattering intensity was recorded on an AVIEX170x170 mm CCD detector over an angular range qmin = 0.01 Å-1 to qmax = 0.5 Å-1. Data averaging and reduction, including preliminary
Rg and I(0) calculations, were carried out using the Foxtrot suite developed at SOLEIL for the SWING beamline. Further analysis was performed with PRIMUS 234 where radius of
gyration values were determined fulfilling the condition qR(g)<1.3. Particle distance distribution function p(r) analysis was carried out using iterative cycles of GNOM 235 using
the Guinier R(g) values as guide for refinement of Dmax. Ab initio 3-D shape reconstruction
was then performed, without the imposition of symmetry restraints for hCCS and hCCS- SOD1 and with an enforced two-fold symmetry axis for SOD1, using the GASBOR web server (EMBL Hamburg) 203. 20 models were averaged with DAMAVER 236 and the
resulting structures compared in PyMOL 237.
9.11 Rigid body modelling
The published crystal structure of hCCS domain II 195 was used as a the structural model
for the hCCS amino acid region 88 – 232 (PDB ID: 1DO5). The structure of region 12 – 69 was inferred from the NMR model of this domain (PDB ID: 2CRL). The secondary structure of the short α-helix found in domain III, amino acids 250 – 258, was inferred using I-TASSER 238. Terminal and linker regions 1 – 11 (with the addition of two extra
amino acids remaining from TEV cleavage of the hexa-histidine tag), 77 – 87, 233 – 249 and 259 – 274 are predicted to be unstructured.
The hCCS homodimer was modelled using BUNCH 204 to generate 25 hCCS models
constrained by the position of the SOD1-like domain II that forms the interface between two CCS monomers. The hCCS-SOD1 heterodimer was modelled using CORAL, a variant of BUNCH that facilitates analysis of protein complexes. In both cases, unrestrained movement of hCCS domain I and III together with the flexible linker regions was permitted. BUNCH and CORAL only generate Cα-atom positions for flexible structural
segments such as linkers and peptide terminal ends. For the purposes of visualisation the protein backbone and sidechain positions of these flexible regions were reconstructed from their Cα traces using the SABBAC web server 239. The scattering profile of each
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