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. Author manuscript; available in PMC: 2019 Nov 1.
Published in final edited form as: J Struct Biol. 2018 Aug 30;204(2):250–260. doi: 10.1016/j.jsb.2018.08.016

Figure 2.

Figure 2.

Biophysical characterization of Sec13/31ΔC compared to wt Sec13/31. a) Distributions of hydrodynamic radii obtained from dynamic light scattering on Sec13/31ΔC heterotetramers in buffers with 10mM, 50mM, 250mM, 450mM, and 700mM concentrations of KOAc. wt Sec13/31 heterotetramers form cages with a diameter of 60nm at 700mM concentration of KOAc. As the graph indicates, the peak of the hydrodynamic radii distribution shifts to a bigger radius as the salt concentration reaches 700mM. The peak of the curve corresponding to 700mM actually happens at 33.7nm which is smaller than the expected value of 60nm. This is in part due to the high ionic concentration medium which reduces the measured hydrodynamic radius indirectly. b) Exemplar negative-staining image of COPII cages made of Sec13/31ΔC and formed in 700mM KOAC buffer. c) Assessing the thermodynamic stability of edges and cages made of wt Sec13/31 and Sec13/31ΔC. Edges and cages are represented by solid lines and dashed lines, respectively, and the wt and the construct are represented by black and red curves, respectively. The cages have an additional minimum in the curve compared to the edges at a temperature of 65 °C for the wt and 62 °C for the ΔC construct. This difference between the melting behavior of cages and edges is attributed to the structural difference between the two structures, namely, the β-propeller contacts that are essential for cage assembly. Although the difference between the wt and the construct for this minimum is small, the difference between the two for the first minimum is around 10 °C. This minimum is attributed to the edge domains that are not involved in the β-propeller contacts at the center of the vertices such as the flexible ACE1 domain. d) Distribution of particle sizes for wt Sec13/31 and Sec13/31ΔC in cage-assembly buffer. According to literature, the sedimentation coefficient of cuboctahedrons is around 60 S. Both histograms show an abundance of particles at that size, but the ΔC construct is more constrained and homogenous in terms of particle size.