XB-IMG-145724
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Figure 4. Crystal structure and solution behaviour of Norrin–Fz4CRD complex.(A) Ribbon representation of Norrin (magenta and pink) in a 2:2:2 complex with Fz4CRD (cyan and pale cyan) and SOS (green). (B) SEC-MALS analyses. The profile of molecular weight (left ordinate axis) and differential refractive index (right ordinate axis) are shown as thick and thin lines, respectively. SDS-PAGE (Inset) shows Norrin in complex with Fz4CRD (triplet band for glycosylated Fz4CRD, marked as green circles, represents glycosylation heterogeneity). (C) SAXS experiments. Experimental scattering data (black circles) and calculated scattering patterns (coloured lines) are shown to a maximal momentum transfer of q = 0.35 Å−1. Individual data: fit pairs are displaced along an arbitrary y axis to allow for better visualization. Bottom curve: Norrin–Fz4CRD 1:2 complex crystal structure (red line). Middle curve: Norrin–Fz4CRD 2:2 complex crystal structure (blue line). Top curve: modelled Norrin–Fz4CRD 2:2 complex crystal structure (missing regions for Norrin and Fz4CRD N- and C-termini are modeled into the crystal complex structure; green line). Structural models are shown in cartoon representation. The bottom left inset shows the experimental (black circles) and calculated (coloured lines) Guinier region. The bottom right inset shows the experimental (black circles) and calculated (coloured lines) pair distance distribution P(r) curves.DOI:
http://dx.doi.org/10.7554/eLife.06554.014Figure 4—figure supplement 1. Protein complex production and structural properties of Norrin–Fz4CRD complex.(A) Norrin forms a stable complex with Fz4CRD in solution. SEC elution profiles and SDS-PAGE under reducing condition are presented. SEC fractions analysed by SDS-PAGE are marked as red lines for Norrin–Fz4CRD complex and cyan lines for uncomplexed Fz4CRD. (B) The crystal structure of methylated Norrin (pink and magenta)–Fz4CRD (cyan) forms a 2:1 complex, which loses one molecule of Fz4CRD during crystal lattice formation. The N-linked glycans are coloured in green. (C) Dimethylated lysine (MLY) residues on the protein surface of methylated Norrin–Fz4CRD are shown as sphere models. Close-up view of sigmaA-weighted 2|FO| − |FC| electron density maps for MLY102 and MLY104 are contoured at 1.0 σ as blue meshes. (D) The sigmaA-weighted |FO| − |FC| electron density maps were calculated with omission of the SOS molecules from the refined model, followed by several cycles of refinement and contoured at 4 σ level as green meshes. (E) Methylated Norrin (yellow)–Fz4CRD (cyan) was superposed onto the Norrin (magenta)–Fz4CRD (blue)–SOS (wheat) complex using Norrin as a reference. (F) The overall sigmaA-weighted 2|FO| − |FC| electron density map after refinement is contoured at 1.0 σ level as blue meshes for the complex of methylated Norrin–Fz4CRD (stick model).DOI:
http://dx.doi.org/10.7554/eLife.06554.015 Image published in: Chang TH et al. (2015) © 2015, Chang et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |